Compositions and Methods For Treating Heart Failure

ABSTRACT

The present invention provides compositions and methods for the following: preventing and treating heart failure; preventing heart failure in a patient with a pre-heart failure condition; treating and preventing heart failure with ischemic and non-ischemic causes; treating and preventing heart failure in a subject status post myocardial infarction; reversing damage to the heart following myocardial infarction; by administering to a subject an effective amount of an adrenergic beta-agonist either alone or in combination with an effective amount of an adrenergic beta-1 antagonist. FIG.  1  is a bar graph representing relative infarct sizes, as expressed as a percentage of left ventricular circumference.

This application claims the benefit of U.S. provisional applicationsU.S. Ser. No. 60/511,619, filed Oct. 13, 2003 and U.S. Ser. No.60/549,803, filed Mar. 2, 2004, which are hereby incorporated byreference into the subject application in its entirety.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

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A portion of the disclosure of this patent document contains or maycontain material, which is subject to copyright protection. Thecopyright owner has no objection to the photocopy reproduction by anyoneof the patent document or the patent disclosure in exactly the form itappears in the Patent and Trademark Office patent file or records, butotherwise reserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION

Heart failure is a leading cause of mortality and morbidity worldwide.In the United States it affects nearly 5 million people and is the onlymajor cardiovascular disorder on the rise. It is estimated that 400,000to 700,000 new cases of heart failure are diagnosed each year in theU.S. and the number of deaths in the U.S. attributable to this conditionhas more than doubled since 1979, currently averaging 250,000 annually.(Heart Failure Association of America). Less than 50 percent of patientssurvive five years after their initial diagnosis of heart failure, andless than 25 percent are alive 10 years after their initial diagnosis.(Heart Failure Association of America). In the more severe cases ofheart failure (New York Heart Association class IV), the 2-yearmortality rate is over 50% (Braunwald, E. B., Heart Disease, 4th ed.(Philadelphia: W.B. Saunders Co., 1992)). Although heart failure affectspeople of all ages, the risk of heart failure increases with age and ismost common among older people. Accordingly, the number of people livingwith heart failure is expected to increase significantly as the elderlypopulation grows over the next few decades. The causes of heart failurehave been linked to various disorders including coronary artery disease,past myocardial infarction, hypertension, abnormal heart valves,cardiomyopathy or myocarditis, congenital heart disease, severe lungdisease, diabetes, severe anemia, hyperthyroidism, arrhythmia ordysrhythmia.

Heart failure, also called congestive heart failure (CHF), is commonlycharacterized by decreased cardiac output, decreased cardiaccontractility, abnormal diastolic compliance, reduced stroke volume, andpulmonary congestion. The clinical manifestations of heart failurereflect a decrease in the myocardial contractile state and a reductionin cardiac output. Apart from deficiencies in cardiac contractility, theCHF disease state may arise from left ventricular failure, rightventricular failure, biventricular failure, systolic dysfunction,diastolic dysfunction, and pulmonary effects. A progressive decrease inthe contractile function of cardiac muscle, associated with heartdisease, often leads to hypoperfusion of critical organs.

For example, in CHF, plasma volume may increase, causing fluid toaccumulate in the lungs, abdominal organs, and peripheral tissues (Beersand Berkow, eds., The Merck Manual of Diagnosis and Therapy, 17th ed.(Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 1682-88).Cardiac arrhythmia, another common feature of heart failure, results inmany of the deaths associated with the disease.

Although symptoms and effects within each category may differ fromindividual to individual, there are two main categories of heartfailure, systolic heart failure and diastolic heart failure. Systolicheart failure is characterized by a decrease in the heart's ability tocontract with sufficient force, resulting in the heart's inability topush enough blood into circulation. In comparison, diastolic failure ischaracterized by a stiffening of the heart muscle. This decrease in theheart's ability to relax, results in the heart's failure to properlyfill with blood during the resting period between each beat.

Drug treatment for heart failure primarily involves diuretics, ACEinhibitors, digoxin (also called digitalis), and beta-blockers. In mildcases, thiazide diuretics, such as hydrochlorothiazide at 25-50 mg/dayor chlorothiazide at 250-500 mg/day, are useful. However, supplementalpotassium chloride is generally needed, since chronic diuresis causeshypokalemis alkalosis. Moreover, thiazide diuretics usually are noteffective in patients with advanced symptoms of Heart failure. Typicaldoses of ACE inhibitors include captopril at 25-50 mg/day and quinaprilat 10 mg/day. Numerous side effects are possible, including decreasedblood pressure, renal insufficiency, potassium retention, and coughing.A more indirect component of heart failure management includes therecognition and control of factors that may be causing increased cardiacdemands or adversely affecting myocardial function (e.g., hypertension,anemia, excess salt intake, excess alcohol, arrhythmias, thyrotoxicosis,fever, increased ambient temperature, or pulmonary emboli) (Beers andBerkow, eds., The Merck Manual of Diagnosis and Therapy, 17th ed.(Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 1688-91).In view of the foregoing, many of the current methods available fortreating heart failure produce negative side-effects and/or treat heartfailure only indirectly. Accordingly, there currently exists a need fornew and better methods for improving the survival of patients with heartfailure.

Myocardial infarction (irreversible damage to heart tissue, often due toheart attack) is a common life-threatening event that may cause suddendeath or heart failure. The ventricular dysfunction that arises aftermyocardial infarction results, primarily, from a massive loss ofcardiomyocytes and gradual replacement of damaged cardiomyocytes withfibrotic non-contractile (scar) tissue. In most cases, the loss ofcardiomyocytes after myocardial infarction is irreversible. Indeed, itis widely accepted that the proliferative (and, therefore, theregenerative) potential of adult mammalian cardiomyocytes is quitelimited (Rumyantsev and Carlson, Growth and Hyperplasia of CardiacMuscle Cells (New York: Harwood Academic Publishers, 1991)), althoughthis view has recently been challenged (Leri et al., Mol. Cell.Cardiol., 3:385-90, 2000; Kajstura et al., Am. J. Pathol., 156:813-19,2000; Beltrami et al., N. Engl. J. Med., 344(23):1750-57, 2001).

Despite considerable advances in the diagnosis and treatment of heartdisease, cardiac damage and dysfunction relating to myocardialinfarction are still among the major cardiovascular disorders.Accordingly, it remains a major therapeutic challenge to find neweffective approaches to improve cardiac function after myocardialinfarction.

Beta-1 and beta-2 adrenergic receptors are expressed in many organs ofthe body including the heart, lungs, and vascular tissue. Thesereceptors mediate the actions of adrenaline and noradrenaline, as wellas various synthetic agonists. In the heart, these receptors regulateheart rate and pumping function; in the lungs, they regulate bronchialtone; and in the vasculature, they regulate vascular tone. Beta-1receptors are instrumental in regulating heart rate, while beta-2receptors play an important role in regulating smooth muscle function.

Beta adrenergic blocking drugs (beta-blockers or beta-antagonists) wereintroduced in the early 1960s. They are commonly used to treathypertension, congestive heart failure, arrhythmias, and angina, and arefrequently used to prevent heart attacks in high-risk patients. Betablockers may also be given to patients who have suffered a heart attack,in order to lessen oxygen consumption by the damaged heart muscle, andprevent sudden death. Beta-blockers slow the nerve impulses travelingthrough heart tissue by blocking the effects of adrenaline on betareceptors. Beta blockers also block the impulses that cause arrhythmia.Beta-blockers can be non-selective or selective for either beta-1 orbeta-2 receptors. For example, Metoprolol, a frequently usedbeta-blocker, is a selective adrenergic beta-1 blocker. Metoprololinhibits the agonistic effect of catecholamines (compounds which arereleased during physical and mental stress) on the heart. Thus,metoprolol reduces the increase in heart rate, cardiac output, cardiaccontractility, and blood pressure produced by an acute increase incatecholamines. Adrenergic beta antagonists include, but are not limitedto acebutolol, alprenolol, amosulalol arotinolol, atenolol, befunolol,betaxolol, bevantolol, bisoprolol, bopindolol, bucindolol, bufetolol,bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol,carazolol, careolol, carvedilol, celiprolol, cetamolol, cloranolol,dilevalol, esmolol, indenolol, labetalol, landiolol, vevobunolol,mepinodolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol,nebivolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol,practolol, pronethalol, proranolol, sotalol, sulfinalol, talinolol,tertatolol, tilisolol, timolol, toliprolol, and xibenolol.

Adrenergic beta agonists include, but are not limited to albuterol,bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline,denopamine, dioxethedrine, dopexamine, ephedrine, epinephrine,etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline,ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol,methoxyphenamine, oxyfedrine, pirbuterol, prenalterol, procaterol,protkylol, reproterol, rimiterol, ritodrine, salmeterol, soterenol,terbutaline, tretoquinol, tulobuterol, and xamoterol. Selectiveadrenergic beta-2 receptor agonists mimic the effects of adrenaline andnoradrenaline, and, therefore, can function as vasodilators. Beta-2receptor agonists are traditionally used to relieve bronchiospasm in thetreatment of respiratory diseases, such as asthma or chronic obstructivepulmonary disease, and are particularly useful in the treatment ofasthma symptoms caused by bronchial constriction, including chesttightness, coughing, and wheezing. Clenbuterol is a long-acting beta2-adrenergic agonist used in the treatment of pulmonary disorders.

Congestive heart failure (“CHF”) is a progressive pathophysiologiccondition where cardiac function is impaired to a degree that the heartis unable to generate output sufficient to meet the metabolicrequirements of the tissues and organs of the body. After initialcardiac injury, in order to compensate for the shortfall in output,myocardial workload increases, as does overall heart mass and size. Theresulting condition of cardiac hypertrophy eventually leads to furtherventricular dysfunction and heart failure. This maladaptive process iscalled cardiac remodeling.

CHF affects 4.7 million patients in the United States and is responsiblefor approximately one million hospitalizations and 300,000 deathsannually (americanheart.org/statistics). The total annual costsassociated with this disorder have been estimated to exceed $22 billion(O'Connel J B, Bristow M R. Economic impact of heart failure in theUnited States: Time for a different approach. Heart Lung Transplant1993; S107-S112.). When the disease enters its terminal phase, the onlycure is heart transplantation. It is estimated that 15,000 patientswould benefit from such a procedure. Unfortunately, due to a shortage ofdonor hearts, only 2,000 heart transplants are performed in the UnitedStates annually. (http://www.unos.org/data/). A need therefore existsfor effective, non-transplant treatment for CHF.

Symptoms of CHF include fatigue, dyspnea and fluid retention in thelungs and extremities. Patients with CHF have reduced exercise capacity,also referred to as “exercise intolerance.” As CHF progressively becomesmore severe, patients are unable to perform basic activities of dailylife. Improving exercise capacity, and concomitantly quality of life, istherefore a primary goal in the management of CHF. The methods oftreatment that are the subject of the present invention meet this need.

Reduced cardiac output has long been viewed as the cause of exerciseintolerance. Prior art medical strategies for treating CHF havetherefore focused on pharmacotherapy to improve hemodynamic function bylowering blood pressure (e.g., with vasodilators), decreasing fluidbuildup (e.g., through use of diuretics) and increasing stroke volume(e.g., with digitalis preparations).

A variant on this hemodynamic-focused paradigm has been suggested. In areview article, Clark et al. (Clark A L, Poole-Wilson P A, Coats A J S.Exercise limitation in chronic heart failure: The central role of theperiphery. J Am Col Cardiol 1996; 28:1092-1102) state that hemodynamicfunction is poorly related to exercise capacity and symptoms in CHF;instead, they state that abnormalities in the peripheral skeletalmusculature are an important determinant of shortness of breath, fatigueand exercise limitation in CHF. Patients with CHF have been shown tohave decreased muscle mass. However, this alone does not explain themarked abnormalities in skeletal muscle strength and exerciseperformance. Several human studies have reported abnormal biochemicaland histological features in skeletal muscle biopsies of patients withCHF (Dunnigan A, Staley N A, Smith S A et al. Cardiac and skeletalmuscle abnormalities in cardiomyopathy: comparison of patients withventricular tachycardiac or congestive heart failure. J Am Col Cardiol1987; 10:608-18; Lipkin D, Jones D, Round J, Poole-Wilson P.Abnormalities of skeletal muscle in patients with chronic heart failure.Int J Cardiology 1988; 18:187-95; Mancini D M, Coyle E, Coggan A, et al.Contribution of intrinsic skeletal muscle changes to ³¹P NMR skeletalmuscle abnormalities in patients with chronic heart failure. Circulation1989; 80:1338-46; Sullivan M J, Green H J, Cobb F R. Skeletal musclebiochemistry and histology in ambulatory patients with long-term heartfailure. Circulation 1990; 81:518-27). Other human studies have shownabnormal skeletal muscle metabolism with rapid depletion of energystores in CHF (Wiener D H, Fink L I, Maris J, et al. Skeletal musclemetabolism in patients with congestive heart failure: role of reducedmuscle flow. Circulation 1986; 73:1127-36; Massie B M, Conway M, YongeR, et al. ³¹P nuclear magnetic resonance evidence of abnormal skeletalmuscle metabolism in patients with congestive heart failure. Am JCardiol 1987; 60:309-15).

Clark et al. stated that pharmacological treatment focused on skeletalmuscle abnormalities (for example, muscle-bulking agents, such asanabolic steroids and b-2 adrenoreceptor agonists) might improve patientquality of life. To date, no therapies are in use that target theskeletal muscle dysfunction in CHF.

While beta-2 adrenoreceptor agonists are primarily used therapeuticallyas bronchodilators in the treatment of asthma and chronic obstructivebronchitis, these agents are also reported in the medical and scientificliterature as having, to varying degrees, anabolic effects on skeletalmuscle. Of the beta-2 adrenoreceptor agonists, clenbuterol is known tohave the most potent anabolic effect on skeletal muscle (Carter W J,Lynch M E. Comparison of the effects of salbutamol and clenbuterol onskeletal muscle mass and carcass composition in senescent rats.Metabolism 1994; 43:1119-1125). This anabolic effect may be related toclenbuterol's prolonged elevated serum levels. Oral clenbuterol has beenused extensively by athletes to enhance muscle size and strength(Muscling in on clenbuterol. Lancet 1992; 340:403; Beckett A H.Clenbuterol and sport (letter). Lancet 1992; 340:1165; Clenbuterol: amedal in tablet form? (letter). Br J Sp Med 1993; 27:141).

Researchers in England have investigated possible use of beta-2adrenoreceptor agonists to improve skeletal muscle functional capacityin humans. Two published studies involved participants in goodcardiovascular health. In the first, Martineau et al. reported thattwice-daily administration of sustained-release salbutamol (Volmax, 8mg) increased voluntary muscle strength in healthy men (Martineau L,Horan M, Rothwell N J, Little R A Salbutamol a β₂-adrenoreceptoragonist, increases skeletal muscle strength in young men. Clin Sci 1992;83:615-621). Maltin et al. reported that post-operative administrationof clenbuterol to males with medial meniscus injury resulted in morerapid rehabilitation of strength in knee extensor muscles and concludedthat beta-adrenoreceptor agonists have therapeutic potential inameliorating muscle-wasting conditions in man (Maltin C A, Delday M I,Watson J S, Hyas S D, Nevison I M, Ritchie I K, Gibson P H. Clenbuterol,a beta-adrenoreceptor agonist, increases relative muscle strength inorthopaedic patients. Clin Sci 1993; 84:651-654). Use of clenbuterol,alone and in combination with beta-blockers, was later stated in threeUS patents, U.S. Pat. Nos. 5,530,029, 5,541,188 and 5,552,442, eachissued to Maltin. These patents teach the use of clenbuterol as atreatment for muscle wasting conditions related to primary musculardisease, neuromuscular abnormalities, generalized catabolic diseasestates, such as cancer and acquired immune deficiency syndrome. TheMaltin patents neither disclose nor suggest the use of clenbuterol forthe skeletal muscle abnormalities of CHF.

The first and only study to date utilizing a beta-2 adrenoreceptoragonist to improve skeletal muscle function in CHF was reported byHarrington et al. (Harrington D, Chua T P, Coats A J S. The effect ofsalbutamol on skeletal muscle in chronic heart failure. Int J Cardiol2000; 73:257-265). After three weeks of treatment with salbutamol (8 mgb.i.d.), there was no change in quadriceps bulk, muscle strength orfatigue and no change in exercise capacity or symptom assessment scores(Minnesota Living with Heart Failure Questionnaire). Accordingly, theinvestigators, themselves, described this as “essentially a negativestudy.”

Harrington et al. hypothesized that a more potent anabolic agent, suchas clenbuterol, might have a role in the treatment of chronic heartfailure myopathy. Persons of ordinary skill in cardiology would be quickto dismiss Harrington's suggestion for reasons of risk-benefit,particularly in light of the negative study results with salbutamol, therisk of mortality from arrhythmia when patients with CHF are treatedwith a beta-adrenergic agent, and data associating clenbuterol use withsignificant myocyte necrosis. More particularly, the risk of suddencardiac death from arrhythmia in the overall adult population is 0.1-0.2percent per year. In patients with CHF with reduced ejection fraction,the risk of sudden death from arrhythmia is 100 times greater (MyerburgR J. Kessler K M. Castellanos A. Structure, function, andtime-dependence of risk. Circulation 1992. 85(1 Suppl):I2-10).Consequently, the administration of any pharmacological agent that mightincrease the risk of arrhythmia in a heart failure patient is avoided.Clenbuterol, like all b-2 adrenergic agents, has some b-1 cardiacactivity and is therefore potentially arrhythmogenic in patients withabnormal cardiac function. Additionally, after demonstrating significantmyocyte necrosis in both heart and skeletal muscle in rats treated withclenbuterol, Burniston et al. concluded, “clenbuterol may be damaging tolong health” (Burniston J, N G Y, Clark W. Myotoxic effects ofclenbuterol in the heart and soleus muscle. J App Physiol 2002;93:1824-1832).

No investigators have studied clenbuterol for the treatment of skeletalmyopathy in stable patients with CHF. However, Yacoub et al., havestudied the effect of very high doses of clenbuterol on cardiac musclein patients with advanced CHF supported with a left ventricular assistdevice (“LVAD”) (Yacoub M. A novel strategy to maximize the efficacy ofleft ventricular assist devices as a bridge to recovery. European HeartJounral 2001; 22:534-540; Yacoub M, Tansley P, Birks E, Hipkin M, HardyJ, Bowles C, Banner N, Khaghani A. Interim results of left ventricularassist device combination therapy for inducing clinical and hemodynamicrecovery of end stage dilated cardiomyopathy. Circulation 2002;106(19):II-606 (Abstr); Hon J, Yacoub M. Bridge to Recovery with the Useof Left Ventricular Assist Device and Clenbuterol. Ann Thorac Surg 2003;75:S36-41).

Patients who are critically ill with end stage heart failure sometimesrequire bridging mechanical support until a heart transplant becomesavailable. The LVAD is an implantable mechanical heart pump that unloadsthe left side of the heart and restores systemic blood flow. In theYacoub study, patients with severe end stage non-ischemic cardiomyopathysupported with an LVAD were treated with high doses of clenbuterol withthe goal of promoting recovery of cardiac function, in order to enableLVAD explantation without transplant. Supratherapeutic doses ofclenbuterol—20 times greater than the recommended dose for treatment ofasthma as well as 20 times the dose administered by Maltin et al. topost-operative orthopoedic patients—were administered to target theheart muscle and promote “physiological” myocyte hypertrophy.

Clenbuterol was given in combination with a standard heart failuremedical regimen including a beta-1 selective blockade, ACE inhibitor,angiotensin-1 receptor antagonists and spironolactone. The theoryunderlying this combination therapy was that by first reducinghemodynamic load with the LVAD, “pathological” cardiac hypertrophy couldbe reversed and then replaced with “physiological” hypertrophy ofcardiac muscle induced by clenbuterol.

The majority of patients in the Yacoub study demonstrated markedimprovement in cardiac function sufficient to tolerate LVAD explantationwithout the need for cardiac transplantation. These preliminary resultssuggest a use for clenbuterol in a very limited subset of CHFpatients—those with non-ischemic cardiomyopathy supported with anLVAD—and does not teach or support the use of clenbuterol in thenon-LVAD, CHF population. The Yacoub study participants were protectedfrom the consequences of arrhythmia by the LVAD, which in the event ofcirculatory collapse from arrhythmia, would mechanically support thecirculation.

Clenbuterol hydrochloride is approved for use in the treatment ofbronchial asthma and chronic obstructive bronchitis in Europe where itis manufactured by various manufacturers, including BoehringerIngelheim, which sells the drug under the brand name Spiropent® in bothtablet and liquid forms. Clenbuterol hydrochloride is not currentlyapproved for any human use by the Food and Drug Administration in theUnited States, but has been approved for clinical study by applicantunder an Investigational New Drug application.

According to the March 2000 package insert, Spiropent® iscontraindicated for use in patients with tachycardic arrhythmia. Thereis also a caution for patients with severe coronary artery disease, whoconstitute approximately half of the heart failure population. TheSeptember 1995 Spiropent® Basic Product Information further advises thatSpiropent® should be used only after careful risk-benefit assessment inpatients with severe organic heart or vascular disorders. (Most patientswith symptoms of CHF have severe organic heart disorder.) Thesewarnings, combined with the general background knowledge in thecardiology community that patients suffering from heart failure are atincreased for sudden death due to arrhythmia, would strongly teach thoseskilled in the art away from using clenbuterol in CHF patients notsupported with an LVAD.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treating andpreventing heart failure by administering to a patient a therapeuticallyeffective amount of a beta-2 agonist. The present invention providescompositions and methods for treating and preventing heart failureresulting from ischemic and non-ischemic causes. In one embodiment ofthe invention, the beta-2 agonist is clenbuterol. In another embodiment,the beta-2 agonist is albuterol, formeoterol, levalbuterol,metaproterenol, pirbuterol, salmeterol, or terbutaline. In anotherembodiment, the method of the present invention treats or prevents heartfailure by treating or preventing cardiac arrhythmia. In still anotherembodiment the method of the invention prevents or treats heart failureby treating or preventing tissue degeneration. In another embodiment,the present invention treats or prevents heart failure by treating orpreventing heart tissue degeneration or reversing the effects of heartfailure through normalization of calcium homeostasis. In an embodimentof the invention, the tissue degeneration can result from myocardialinfarction. In an embodiment, the dosage of the beta-2 agonist is about0.01 mg/kg/day to about 2.0 mg/kg/day. The beta-2 agonist clenbuterolcan be administered in a dosage of about 5 mcg/day to 100 mg/day.Preferably, clenbuterol is administered in a dosage of bout 80 mcg/dayto 1.5 mg/day.

The invention further provides compositions and methods for treatingheart failure by administering to a patient a therapeutically effectiveamount of a beta-2 agonist in combination with a therapeuticallyeffective amount of an adrenergic beta-1 antagonist. The beta-2 agonistand the beta-1 antagonist can be administered concurrently, sequentiallyor alternately. In a preferred embodiment, a synergistic therapeuticeffect results from this combination therapy. The beta-2 agonist can beselected from the group consisting of clenbuterol, albuterol,formeoterol, levalbuterol, metaproterenol, pirbuterol, salmeterol, andterbutaline. In a preferred embodiment of the invention, the beta-2agonist is clenbuterol. The beta-1 antagonist can be selected from thegroup consisting of acebutolol, atenolol, betaxolol, bisoprolol,esmolol, and metoprolol. In a preferred embodiment, the beta-1antagonist is metoprolol. In an embodiment, the dosage of the beta-1antagonist is about 15 mg/kg/day to about 300 mg/kg/day. The beta-1antagonist metoprolol can be administered in a dosage of about 01.mcg/day to 500 mg/day. Preferably, metoprolol is administered in adosage of about 5 mg/day to 300 mg/day.

In another embodiment, the method of the present invention treats orprevents heart failure by treating or preventing cardiac arrhythmia. Instill another embodiment the method of the invention prevents or treatsheart failure by treating or preventing tissue degeneration. In oneembodiment of the invention, heart failure can result from both ischemicand non-ischemic causes. In another embodiment of the invention, thetissue degeneration can result from myocardial infarction.

The invention further provides a method for preventing heart failure ina subject with a pre-heart failure condition, comprising administeringto the subject a therapeutically effective amount of an adrenergicbeta-2 agonist either alone or in combination with a therapeuticallyeffective amount of an adrenergic beta-1 antagonist. When administeredin combination, the beta-2 agonist and the beta-1 antagonist can beadministered concurrently, sequentially or alternately. In a preferredembodiment a synergistic therapeutic effect results from thiscombination therapy. The beta-2 agonist can be selected from the groupconsisting of clenbuterol, albuterol, formeoterol, levalbuterol,metaproterenol, pirbuterol, salmeterol, and terbutaline. The dosages ofthe beta-2 agonist can be administered as previously described above. Ina preferred embodiment of the invention, the beta-2 agonist isclenbuterol. The beta-1 antagonist can be selected from the groupconsisting of acebutolol, atenolol, betaxolol, bisoprolol, esmolol, andmetoprolol. The dosages of the beta-1 antagonist can be administered aspreviously described above. In a preferred embodiment, the beta-1antagonist is metoprolol.

In another embodiment, the method of the present invention preventsheart failure in a subject with a pre-heart failure condition bytreating or preventing cardiac arrhythmia. In still another embodimentthe method of the invention prevents heart failure in a subject with apre-heart failure condition by treating or preventing tissuedegeneration. In an embodiment of the invention, the tissue degenerationcan result from myocardial infarction.

In another embodiment, the method of the present invention preventsheart failure in a subject post myocardial infarction by preventingcardiac arrhythmia. In still another embodiment the method of theinvention prevents heart failure in a subject with a pre-heart failurecondition by treating or preventing tissue degeneration. In anembodiment of the invention, the tissue degeneration can result frommyocardial infarction.

The invention further provides a method for preventing heart failure ina patient status post myocardial infarction, comprising administering tothe subject a therapeutically effective amount of an adrenergic beta-2agonist either alone or in combination with a therapeutically effectiveamount of an adrenergic beta-1 antagonist. When administered incombination, the beta-2 agonist and the beta-1 antagonist can beadministered concurrently, sequentially or alternately. In a preferredembodiment a synergistic therapeutic effect results from thiscombination therapy. The beta-2 agonist can be selected from the groupconsisting of clenbuterol, albuterol, formeoterol, levalbuterol,metaproterenol, pirbuterol, salmeterol, and terbutaline. The dosages ofthe beta-2 agonist can be administered as previously described above. Ina preferred embodiment of the invention, the beta-2 agonist isclenbuterol. The beta-1 antagonist can be selected from the groupconsisting of acebutolol, atenolol, betaxolol, bisoprolol, esmolol, andmetoprolol. The dosages of the beta-1 antagonist can be administered aspreviously described above. In a preferred embodiment, the beta-1antagonist is metoprolol.

In another embodiment, the method of the present invention preventsheart failure in a patient status post myocardial infarction by treatingor preventing cardiac arrhythmia. In still another embodiment the methodof the invention prevents heart failure in a subject with a pre-heartfailure condition by treating or preventing tissue degeneration. In anembodiment of the invention, the tissue degeneration can result frommyocardial infarction.

The present invention additionally provides for a method of treating orpreventing heart failure in a subject, comprising administering to thesubject an amount of an adrenergic beta-2 agonist effective to treat orprevent the heart failure, in combination with an amount of anadrenergic beta-1 antagonist effective to reduce the toxicity of theadrenergic beta-2 agonist. In a preferred embodiment of the invention,the adrenergic beta-2 agonist is clenbuterol and the adrenergic beta-1antagonist is metoprolol. In another embodiment of the invention thebeta-2 agonist can be selected from the group consisting of clenbuterol,albuterol, formeoterol, levalbuterol, metaproterenol, pirbuterol,salmeterol, and terbutaline. The adrenergic beta-1 antagonist can beselected from the group consisting of acebutolol, atenolol, betaxolol,bisoprolol, esmolol, and metoprolol. The beta-2 agonist and the beta-1antagonist can be administered concurrently, sequentially oralternately. Preferably, a synergistic therapeutic effect results fromthis combination therapy.

The present invention further provides a method for reversing damage tothe heart following myocardial infarction using a combination of anadrenergic beta-1 antagonist and an adrenergic beta-2 agonist. In apreferred embodiment of the invention, the adrenergic beta-2 agonist isclenbuterol and the adrenergic beta-1 antagonist is metoprolol. Inanother embodiment of the invention the beta-2 agonist can be selectedfrom the group consisting of clenbuterol, albuterol, formeoterol,levalbuterol, metaproterenol, pirbuterol, salmeterol, and terbutaline.The adrenergic beta-1 antagonist can be selected from the groupconsisting of acebutolol, atenolol, betaxolol, bisoprolol, esmolol, andmetoprolol. The beta-2 agonist and the beta-1 antagonist can beadministered concurrently, sequentially or alternately. Preferably, asynergistic therapeutic effect results from this combination therapy.

The present invention is additionally encompasses kits for use intreating or preventing heart failure and/or reversing damage to theheart following a heart attack comprising administering a combination ofan adrenergic beta-1 antagonist and an adrenergic beta-2 agonist. In apreferred embodiment of the invention, the adrenergic beta-2 agonist isclenbuterol and the adrenergic beta-1 antagonist is metoprolol. Inanother embodiment of the invention the beta-2 agonist can be selectedfrom the group consisting of clenbuterol, albuterol, formeoterol,levalbuterol, metaproterenol, pirbuterol, salmeterol, and terbutaline.The adrenergic beta-1 antagonist can be selected from the groupconsisting of acebutolol, atenolol, betaxolol, bisoprolol, esmolol, andmetoprolol. The beta-2 agonist and the beta-1 antagonist can beadministered concurrently, sequentially or alternately. Preferably, asynergistic therapeutic effect results from this combination therapy.

Additional aspects of the present invention will be apparent in view ofthe description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph representing relative infarct sizes, as expressedas a percentage of left ventricular circumference. While the LADligation animals had significantly larger infarct sizes than the Shamgroup, there were no differences in the infarct sizes among the LADligation groups.

FIG. 2 is a bar graph representing echocardiographic data, as expressedby fractional shortening (FS) and fractional area change (FAC). Each ofthe LAD ligation groups had a significantly lower FS and FAC than theSham animals. There were no significant changes in FS or FAC between thebaseline and endpoint parameters for any of the groups.

FIG. 3 is a bar graph representing Hemodynamic data after 12 weeks offollow-up. For left ventricular end-diastolic pressure (LVEDP), therewere significantly higher diameters in the HF, Clen, and Clen+Metogroups versus the Sham group. However, metoprolol-treated rats had alower LVEDP than the control HF group. For the maximum dP/dt, control HFand Clen rats had significantly lower values than Sham rats. There wasno difference between the Meto or Clen+Meto group and the Sham group.

FIG. 4 is a graphical representation of ex vivo end-diastolicpressure-volume-relationship (EDPVR) tracings, after normalization of LVvolumes for differences in heart weights. Clenbuterol-treated rats wereshifted to the left vs. both control HF and Meto rats and were nodifferent from Sham rats. In contrast, HF, Meto, and Clen+Meto rats hadhigher passive LV volumes than Sham rats.

FIG. 5A shows pictures representing photomicrographs representative ofimmunohistochemistry staining patterns for the apoptosis marker, TUNEL.

FIG. 5B shows pictures representing photomicrographs representative ofimmunohistochemistry staining patterns for the apoptosis marker, 8-oxoG.

FIG. 5C shows pictures representing photomicrographs representative ofimmunohistochemistry staining patterns for the apoptosis marker, OGG1.

FIG. 5D shows pictures representing photomicrographs representative ofimmunohistochemistry staining patterns for the apoptosis marker, MYH.

FIG. 6. Calcium-handling protein expression levels of the ryanodinereceptor (RyR) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA_(2a)),expressed relative to tubulin expression levels. The mean opticaldensity units of RyR and SERCA^(2a) levels are decreased in the HFgroup, as compared to the Sham group. Clen-treated rats hadsignificantly increased levels of RyR and SERCA_(2a) vs. control HFrats. Representative autoradiographs are depicted for RyR andSERCA_(2a), with the corresponding tubulin blotting signals.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used in this example: LVAD, leftventricular assist device; HF, heart failure; LV, left ventricular; RyR,ryanodine receptor; SERCA2a, sarcoplasmic reticulum calcium-ATPase; LAD,left anterior descending artery; Clen, clenbuterol; Meto, metoprolol;Clen+Meto, clenbuterol and metoprolol; LVEDD, left ventricularend-diastolic diameter; LVEDA, left ventricular end-diastolic area;LVESD, left ventricular end-systolic diameter; LVESA, left ventricularend-systolic area; FS, fraction shortening; FAC, fractional area change;EDPVR, end-diastolic pressure-volume relationships; LVEDP, leftventricular end-diastolic pressure; LVSP, left ventricular systolicpressure; MAoP, mean aortic pressure; dP/dtmax, maximum left ventriculardP/dt; dP/dtmin, minimum left ventricular dP/dt; TUNEL, terminaldeoxynucleotidyltransferase end labeling; ANOVA, analysis of variance;SD, standard deviation.

There are no therapies available that directly target the abnormalskeletal musculature in congestive heart failure. The present inventiondiscloses novel methods for administering clenbuterol to patients notsupported with an LVAD with all classes of congestive heart failure,from mild to severe, including acute and chronic heart failuresyndromes, as well as patients with asymptomatic ventricular dysfunctionand in patients after myocardial infarction. Clenbuterol will improveskeletal muscle function, exercise capacity, fatigue, quality of life,as well as cardiac function in patients with CHF.

For purposes of the present invention (except where context specificallyrequires an alternative construction), clenbuterol shall be understoodto mean clenbuterol free base as well as pharmaceutical alternativesthereof containing the same therapeutic moiety (includingpharmaceutically acceptable salts thereof, such as hydrochloride,hydrobromide etc., esters or complexes of the moiety) and includes theindividual active optical isomers thereof (each alone or in non-racemicmixtures thereof), polymorphs and mixtures thereof.

According to the methods of treatment of the present invention,clenbuterol is administered to patients with CHF not supported with anLVAD. Clenbuterol may be administered by a variety of routes ofadministration, both immediate and extended release, including solidoral dosage forms (e.g., tablets, capsules), liquid oral dosage forms,intravenous or intramuscular or subcutaneous injection, topicaladministration or by inhalation. Clenbuterol supplements existingapproved therapies for CHF including angiotensin converting enzymeinhibitors (ACE inhibitors), beta adrenoreceptor blocking agents,digoxin and spironolactone. As such co-therapy using clenbuterol and oneor more of these agents in floating and fixed combinations are alsosuitable. Other medicinal combination therapy using clenbuterol withmembers of these classes and related classes of medicinal agents will beapparent to those of ordinary skill in the art.

The present invention also teaches methods for safely administeringclenbuterol to improve skeletal muscle function in patients with CHF forwhom administration of a beta adrenergic agonist, such as clenbuterol,would otherwise be contraindicated because of the risk of arrhythmia. Asdiscussed above, patients with CHF and reduced ventricular function havea 100-fold increased risk of sudden death from arrhythmia. Clenbuterolpossesses some beta-1 adrenergic activity and is potentiallyarryhthmogenic for patients with CHF.

According to the risk-stratified approach of the present invention,patients at highest risk for arrhythmia, such as those with ischemiccardiomyopathy or with a past history of arrhythmia, are administeredclenbuterol in combination with a beta-1 selective blocker and afterimplantable Cardioverter-Defibrillator (“ICD”) implantation. The ICD isan electrical device used in patients at high risk for arrhythmia or inpatients who have suffered an episode of significant arrhythmia such asventricular tachycardia or sudden cardiac death. It detects seriousarrhythmia and delivers therapy to restore normal rhythm. Patientspreviously taking a non-selective beta-blocking agent are switched to abeta-1 selective blocking agent. The use of a beta-1 selective blockingagent such as metoprolol or bisoprolol diminishes the cardiac effects ofclenbuterol (through the beta-1 receptor) without diminishing its effecton skeletal muscle (through the beta-2 receptor).

Patients at lower risk for arrhythmia are administered clenbuterol incombination with a beta-1 selective blocker and without ICD. Patientsconsidered at lowest risk for arrhythmia, such as those with mildventricular dysfunction and in whom a beta-1 selective blocker wascontraindicated (such as in obstructive airways disease and severeperipheral vascular disease), are administered clenbuterol without abeta-1 selective blocker or ICD. In the present invention, oneembodiment is where the patient or subject is not supported by an LVAD.

Patients treated according to the method of treatment of the presentinvention are dosed over a broad range. Initially, patients receive astarting daily dose of about 40 mcg and are gradually up-titrated astolerated to a maximal daily dose of about 4 mg. Up-titration occurs ona weekly basis in the absence of significant adverse effects.

The goal of the present invention is to improve skeletal musclefunction, exercise capacity, quality of life and cardiac function inpatients with CHF not supported with an LVAD. Objective measures ofthese parameters are well-known to persons of ordinary skill in the artand include the following. Skeletal muscle function is measured usingstandard tests of isometric muscle strength and fatigue and can beexpressed as Maximal Strength (normalized for muscle cross-sectionalarea) and the Static Fatigue Index. Exercise capacity in patients withCHF is measured by cardiopulmonary exercise testing and is expressed asPeak Oxygen Consumption, Peak Work and Exercise Duration. Quality oflife is measured using standard questionnaires such as the MinnesotaLiving with Heart Failure (MLHF) Questionnaire (Rector T S, Kubo S H,Cohn J N. Patients' self-assessment of their congestive heart failure.Part 2: Content, reliability and validity of a new measure, theMinnesota Living with Heart Failure questionnaire. Heart Failure 1987;October/November: 198-209.). Cardiac function is measured byechocardiography, or by cardiac nuclear scanning techniques (MUGA scan)and is expressed as Ejection Fraction.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20 percent up or down (higher or lower).

As used herein, the word “or” means any one member of a particular listand also includes any combination of members of that list.

The present invention encompasses methods for treating and preventingheart failure in a subject by administering to the subject atherapeutically effective amount of an adrenergic beta-2 agonist eitheralone or in combination with an adrenergic beta-1 antagonist.

The terms “congestive heart failure (CHF)”, “chronic heart failure”,“acute heart failure”, and “heart failure” are used interchangeablyherein and refer to any condition characterized by abnormally lowcardiac output in which the heart is unable to pump blood at an adequaterate or in adequate volume. When the heart is unable to adequately pumpblood to the rest of the body, or when one or more of the heart valvesbecomes stenotic or otherwise incompetent, blood can “back up” into thelungs, causing the lungs to become congested with fluid. If thisbackward flow occurs over an extended period of time, heart failure canresult. Typical symptoms of heart failure include shortness of breath(dyspnea), fatigue, weakness, difficulty breathing when lying flat, andswelling of the legs, ankles or abdomen (edema). Causes of heart failureare related to various disorders including coronary artery disease,systemic hypertension, cardiomyopathy or myocarditis, congenital heartdisease, abnormal heart valves or valvular heart disease, severe lungdisease, diabetes severe anemia hyperthyroidism, arrhythmia ordysrhythmia and myocardial infarction. The three cardinal signs ofcongestive heart failure are: cardiomegaly (enlarged heart), tachypnea(rapid breathing; occurs in the case of left side failure) andhepatomegaly (enlarged liver; occurs in the case of right side failure).

Treating heart failure, as used herein, refers to treating any one ormore of the conditions underlying heart failure, including, withoutlimitation, decreased cardiac contractility, abnormal diastoliccompliance, reduced stroke volume, pulmonary congestion, and decreasedcardiac output. As further used herein, “oxygen-wasting effects”include, without limitation, symptoms and signs of congestion due toincreased ventricular filling pressures, and fatigue associated with lowcardiac output. As used herein, preventing heart failure includespreventing the initiation of heart failure, delaying the initiation ofheart failure, preventing the progression or advancement of heartfailure, slowing the progression or advancement of heart failure,delaying the progression or advancement of heart failure, and reversingthe progression of heart failure from an advanced to a less advancedstage.

In one embodiment of the invention, heart failure is treated in asubject in need of treatment by administering to the subject atherapeutically effective amount of an adrenergic beta-2 agonisteffective to treat the heart failure. The subject is preferably a mammal(e.g., humans, domestic animals, and commercial animals, including cows,dogs, monkeys, mice, pigs, and rats), and is most preferably a human.The term “therapeutically effective amount,” or “effective amount” asused herein mean the quantity of the composition according to theinvention which is necessary to prevent, cure, ameliorate or at leastminimize the clinical impairment, symptoms or complications associatedwith heart failure in either a single or multiple dose. The amounts ofadrenergic beta-2 agonist and beta-1 antagonist effective to treat heartfailure will vary depending on the particular factors of each case,including the stage or severity of heart failure, the subject's weight,the subject's condition and the method of administration. The skilledartisan can readily determine these amounts.

Adrenergic beta-1 blockers (antagonists), such as metoprolol, have beenused as essential therapies for heart-failure patients. However, priorto the present invention, adrenergic beta-2 agonists, such asclenbuterol, have traditionally been used to treat asthma. Clenbuterol,in particular, has been approved in the European Union for the treatmentof asthma, and is often administered to athletes to improve performancecapacity. The present invention establishes that adrenergic beta-2agonists such as clenbuterol can also be used to prevent and treatheart-failure patients either alone or in combination with an adrenergicbeta-1 antagonist such as metoprolol. This new therapy will provide aunique strategy to reverse the remodeling of the left ventricle duringheart failure and after myocardial infarction (heart attack).

Metoprolol and clenbuterol target heart failure via differentmechanisms: metoprolol blocks beta-1 receptors and correctsneurohormonal imbalance, while clenbuterol stimulates beta-2 receptorsand rescues myocytes, is anti-apoptotic, and/or improves the function ofcalcium-handling proteins. It is believed that an adrenergic beta-1blocker for use in the present invention will block the possibletoxicity of high-dose adrenergic beta-2 agonist; the adrenergic beta-2agonist is then expected to increase the mass of the heart (and skeletalmuscle) physiologically, and rescue myocytes from apoptosis andnecrosis. Thus, metoprolol and clenbuterol produce unexpectedsynergistic effects in the treatment of heart failure. Furthermore,clenbuterol, when used in combination with metoprolol, may beadministered in amounts lower than would otherwise be expected.

As used herein, “adrenergic beta-2 agonist” refers to adrenergic beta-2agonists and analogues and derivatives thereof, including, for example,natural or synthetic functional variants which have adrenergic beta-2agonist biological activity, as well as fragments of an adrenergicbeta-2 agonist having adrenergic beta-2 agonist biological activity. Asfurther used herein, the term “adrenergic beta-2 agonist biologicalactivity” refers to activity that mimics the effects of adrenaline andnoradrenaline in a subject and which improves myocardial contractilityin a patient having heart failure. Commonly known adrenergic beta-2agonists include, but are not limited to, clenbuterol, albuterol,formeoterol, levalbuterol, metaproterenol, pirbuterol, salmeterol, andterbutaline.

As used herein, “adrenergic beta-1 antagonist” and adrenergic beta-1blocker are used interchangeably and refer to adrenergic beta-1antagonists and analogues and derivatives thereof, including, forexample, natural or synthetic functional variants which have adrenergicbeta-1 antagonist biological activity, as well as fragments of anadrenergic beta-1 antagonist having adrenergic beta-1 antagonistbiological activity. As further used herein, the term “adrenergic beta-1antagonist biological activity” refers to activity that blocks theeffects of adrenaline on beta receptors. Commonly known adrenergicbeta-1 antagonists include, but are not limited to, acebutolol,atenolol, betaxolol, bisoprolol, esmolol, and metoprolol.

Methods of preparing adrenergic beta-2 agonists such as clenbuterol andtheir analogues and derivatives are well known in the art. Clenbuterol,for example, is available from MP Biomedicals, Inc. 1263 S. ChillicotheRd., Aurora, Ohio 44202. Clenbuterol is also commercially availableunder numerous brand names including Spiropent® (Boehinger Ingelheim),Broncodil® (Von Boch I), Broncoterol® (Quimedical PT), Cesbron® (FidelisPT), and Clenbuter® (Biomedica Foscama). Similarly, methods of preparingadrenergic beta-1 antagonists such as metoprolol and their analogues andderivatives are well-known in the art. Metoprolol, in particular, iscommercially available under the brand names Lopressor® (metoprololtartate) manufactured by Novartis Pharmaceuticals Corporation, OneHealth Plaza, East Hanover, N.J. 07936-1080. Generic versions ofLopressor® are also available from Mylan Laboratories Inc., 1500Corporate Drive, Suite 400, Canonsburg, Pa. 15317; and WatsonPharmaceuticals, Inc., 360 Mt. Kemble Ave. Morristown, N.J. 07962.Metoprolol is also commercially available under the brand name ToprolXL®, manufactured by Astra Zeneca, LP. Moreover, both beta-2 agonistsand beta-1 antagonists may be synthesized in accordance with knownorganic chemistry procedures that are readily understood by those ofskill in the art.

In a method of the present invention, an adrenergic beta-2 agonist isadministered to a subject in combination with an adrenergic beta-1agonist, such that a synergistic therapeutic effect is produced. A“synergistic therapeutic effect” refers to a greater-than-additivetherapeutic effect which is produced by a combination of two therapeuticagents, and which exceeds that which would otherwise result fromindividual administration of either therapeutic agent alone. Forinstance, administration of clenbuterol in combination with metoprololunexpectedly results in a synergistic therapeutic effect by providinggreater efficacy than would result from use of either of the therapeuticagents alone. Clenbuterol enhances metoprolol's effects. Therefore,lower doses of one or both of the therapeutic agents may be used intreating heart failure, resulting in increased therapeutic efficacy anddecreased side-effects.

In the method of the present invention, administration of an adrenergicbeta-2 agonist “in combination with” an adrenergic beta-1 antagonistrefers to co-administration of the two therapeutic agents.Co-administration may occur concurrently, sequentially, or alternately.Concurrent co-administration refers to administration of both theadrenergic beta-2 agonist and the adrenergic beta-1 antagonist atessentially the same time. For concurrent co-administration, the coursesof treatment with the adrenergic beta-2 agonist and with the adrenergicbeta-1 antagonist may be run simultaneously. For example, a single,combined formulation, containing both an amount of an adrenergic beta-2agonist and an amount of an adrenergic beta-1 antagonist in physicalassociation with one another, may be administered to the subject. Thesingle, combined formulation may consist of an oral formulation,containing amounts of both the beta-2 agonist and the beta-1 antagonist,which may be orally administered to the subject, or a liquid mixture,containing amounts of both the beta-2 agonist and the beta-1 antagonist,which may be injected into the subject.

It is also provided by the present invention that an amount of theadrenergic beta-2 agonist and an amount of the adrenergic beta-1antagonist may be administered concurrently to a subject, in separate,individual formulations. Accordingly, the method of the presentinvention is not limited to concurrent co-administration of theadrenergic beta-2 agonist and the adrenergic beta-1 antagonist inphysical association with one another.

In the method of the present invention, the adrenergic beta-2 agonistand the adrenergic beta-1 antagonist also may be co-administered to asubject in separate, individual formulations that are spaced out over aperiod of time, so as to obtain the maximum efficacy of the combination.Administration of each therapeutic agent may range in duration from abrief, rapid administration to a continuous perfusion. When spaced outover a period of time, co-administration of the adrenergic beta-2agonist and the adrenergic beta-1 antagonist may be sequential oralternate. For sequential co-administration, one of the therapeuticagents is separately administered, followed by the other. For example, afull course of treatment with the adrenergic beta-2 agonist may becompleted, and then may be followed by a full course of treatment withthe adrenergic beta-1 antagonist. Alternatively, for sequentialco-administration, a full course of treatment with the adrenergic beta-1antagonist may be completed, then followed by a full course of treatmentwith the adrenergic beta-2 agonist. For alternate co-administration,partial courses of treatment with the adrenergic beta-2 agonist may bealternated with partial courses of treatment with the adrenergic beta-1antagonist, until a full treatment of each therapeutic agent has beenadministered.

The therapeutic agents of the present invention (i.e., the adrenergicbeta-2 agonist and the adrenergic beta-1 antagonist, either in separate,individual formulations, or in a single, combined formulation) may beadministered to a human or animal subject by known procedures,including, but not limited to, oral administration, parenteraladministration (e.g., intramuscular, intraperitoneal, intravascular,intravenous, or subcutaneous administration), and transdermaladministration. Preferably, the therapeutic agents of the presentinvention are administered orally or intravenously.

For oral administration, the formulations of the adrenergic beta-2agonist either alone or in combination with the adrenergic beta-1antagonist may be presented as capsules, tablets, powders, granules, oras a suspension. The formulations may have conventional additives, suchas lactose, mannitol, corn starch, or potato starch. The formulationsalso may be presented with binders, such as crystalline cellulose,cellulose derivatives, acacia, corn starch, or gelatins. Additionally,the formulations may be presented with disintegrators, such as cornstarch, potato starch, or sodium carboxymethyl cellulose. Theformulations also may be presented with dibasic calcium phosphateanhydrous or sodium starch glycolate. Finally, the formulations may bepresented with lubricants, such as talc or magnesium stearate.

For parenteral administration, the formulations of the adrenergic beta-2agonist either alone or in combination with the adrenergic beta-1antagonist may be combined with a sterile aqueous solution which ispreferably isotonic with the blood of the subject. Such formulations maybe prepared by dissolving a solid active ingredient in water containingphysiologically-compatible substances, such as sodium chloride, glycine,and the like, and having a buffered pH compatible with physiologicalconditions, so as to produce an aqueous solution, then rendering saidsolution sterile. The formulations may be presented in unit ormulti-dose containers, such as sealed ampules or vials. Moreover, theformulations may be delivered by any mode of injection, including,without limitation, epifascial, intracapsular, intracutaneous,intramuscular, intraorbital, intraperitoneal (particularly in the caseof localized regional therapies), intraspinal, intrasternal,intravascular, intravenous, parenchymatous, or subcutaneous.

For transdermal administration, the formulations of the adrenergicbeta-2 agonist and the adrenergic beta-1 antagonist (whether individualor combined) may be combined with skin penetration enhancers, such aspropylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, and the like, which increase the permeability ofthe skin to the therapeutic agent, and permit the therapeutic agent topenetrate through the skin and into the bloodstream. The therapeuticagent/enhancer compositions also may be further combined with apolymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which may be dissolved in a solvent such asmethylene chloride, evaporated to the desired viscosity, and thenapplied to backing material to provide a patch.

The dose of the adrenergic beta-2 agonist and the adrenergic beta-1antagonist of the present invention may also be released or deliveredfrom an osmotic mini-pump. The release rate from an elementary osmoticmini-pump may be modulated with a microporous, fast-response geldisposed in the release orifice. An osmotic mini-pump would be usefulfor controlling release, or targeting delivery, of the therapeuticagents.

It is within the confines of the present invention that the formulationsof the adrenergic beta-2 agonist either alone or in combination with theadrenergic beta-1 antagonist may be further associated with apharmaceutically-acceptable carrier, thereby comprising a pharmaceuticalcomposition. The pharmaceutically-acceptable carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the composition, and not deleterious to the recipient thereof.Examples of acceptable pharmaceutical carriers include, but are notlimited to, carboxymethyl cellulose, crystalline cellulose, glycerin,gum arabic, lactose, magnesium stearate, methyl cellulose, powders,saline, sodium alginate, sucrose, starch, talc, and water, among others.Formulations of the pharmaceutical composition may conveniently bepresented in unit dosage.

The formulations of the present invention may be prepared by methodswell-known in the pharmaceutical art. For example, the active compoundmay be brought into association with a carrier or diluent, as asuspension or solution. Optionally, one or more accessory ingredients(e.g., buffers, flavoring agents, surface active agents, and the like)also may be added. The choice of carrier will depend upon the route ofadministration. The pharmaceutical composition would be useful foradministering the therapeutic agents of the present invention (i.e., theadrenergic beta-2 agonist and the adrenergic beta-1 antagonist, andtheir analogues and derivatives, either in separate, individualformulations, or in a single, combined formulation) to a subject totreat heart failure. The therapeutic agents are provided in amounts thatare effective to treat or prevent heart failure in the subject. Theseamounts may be readily determined by the skilled artisan.

In the synergistic combination of the present invention, the adrenergicbeta-2 agonist and the adrenergic beta-1 antagonist may be combined in asingle formulation, such that the amount of the adrenergic beta-2agonist is in physical association with the amount of the adrenergicbeta-1 antagonist. This single, combined formulation may consist of anoral formulation, containing amounts of both the adrenergic beta-2agonist and the adrenergic beta-1 antagonist, which may be orallyadministered to the subject, or a liquid mixture, containing amounts ofboth the adrenergic beta-2 agonist and the adrenergic beta-1 antagonist,which may be injected into the subject.

Alternatively, in the synergistic combination of the present invention,a separate, individual formulation of the adrenergic beta-2 agonist maybe combined with a separate, individual formulation of the adrenergicbeta-1 antagonist. For example, an amount of the adrenergic beta-2agonist may be packaged in a vial or unit dose, and an amount of theadrenergic beta-1 antagonist may be packaged in a separate vial or unitdose. A synergistic combination of the adrenergic beta-2 agonist and theadrenergic beta-1 antagonist then may be produced by mixing the contentsof the separate vials or unit doses in vitro. Additionally, asynergistic combination of the adrenergic beta-2 agonist and theadrenergic beta-1 antagonist may be produced in vivo by co-administeringto a subject the contents of the separate vials or unit doses, accordingto the methods described above. Accordingly, the synergistic combinationof the present invention is not limited to a combination in whichamounts of the adrenergic beta-2 agonist and the adrenergic beta-1antagonist are in physical association with one another in a singleformulation.

The synergistic combination of the present invention comprises aneffective therapeutic amount of the adrenergic beta-2 agonist and aneffective therapeutic amount of the adrenergic beta-1 antagonist. Asused herein, an “therapeutically effective amount” of the adrenergicbeta-2 agonist or the adrenergic beta-1 antagonist is an amount of theadrenergic beta-2 agonist or the adrenergic beta-1 antagonist that iseffective to ameliorate or minimize the clinical impairment or symptomsof heart failure in a subject, in either a single or multiple dose. Forexample, the clinical impairment or symptoms of heart failure may beameliorated or minimized by diminishing any pain or discomfort sufferedby the subject; by extending the survival of the subject beyond thatwhich would otherwise be expected in the absence of such treatment; orby inhibiting or preventing the progression of the heart failure, or byreversing the pathologic processes involved in heart failure.

The effective therapeutic amounts of the adrenergic beta-2 agonist andthe adrenergic beta-1 antagonist will vary depending on the particularfactors of each case, including the stage of the heart failure, thesubject's weight, the severity of the subject's condition, and themethod of administration. For example, the beta-2 agonist clenbuterolcan be administered in a dosage of about 5 mcg/day to 100 mg/day.Preferably, clenbuterol is administered in a dosage of bout 80 mcg/dayto 1.5 mg/day.

In a preferred embodiment, the dosage of beta-2 agonist is about 0.01mg/kg/day to about 2.0 mg/kg/day. In one embodiment, albuterol can beadministered in a dosage of about 10 mcg/day to 200 mg/day, andpreferably, is administered in a dosage about 2 mg/day to 32 mg/day.Formoterol can be administered in a dosage of about 1 mcg/day to 200mg/day, and preferably, is administered in a dosage of about 12 mcg/dayto 2 mg/day. Levalbuterol can be administered in a dosage of about 1mcg/day to 200 mg/day, and preferably, is administered in a dosage ofabout 100 mcg/day to 100 mg/day. Metaproterenol can be administered in adosage of about 0.1 mcg/day to 200 mg/day, and preferably, isadministered in a dosage of about 2 mcg/day to 2 mg/day. Pirbuterol canbe administered in a dosage of about 0.1 mcg/day to 200 mg/day, andpreferably, is administered in a dosage of about 2 mcg/day to 2 mg/day.Salmeterol can be administered in a dosage of about 1 mcg/day to about200 mg/day, and preferably, is administered in a dosage of about 2mcg/day to 2 mg/day. Terbutaline can be administered in a dosage ofabout 0.1 mcg/day to 200 mg/day, and preferably, is administered in adosage of about 5 mg to 20 mg/day.

The beta-1 antagonist metoprolol can be administered in a dosage ofabout 01 .mcg/day to 300 mg/day. Preferably, metoprolol is administeredin a dosage of about 5 mg/day to 200 mg/day. Acebutolol can beadministered in a dosage of about 50 mg/day to 5000 mg/day, andpreferably, is administered in a dosage of about 200 mg/day to 1200mg/day. Atenolol can be administered in a dosage of about 1 mg/day to500 mg/day, and preferably, is administered in a dosage of about 25mg/day to 100 mg/day. Betaxolol can be administered in a dosage of about1 mg/day to 100 mg/day, and preferably, is administered in a dosage ofabout 5 mg/day to 20 mg/day. Bisoprolol can be administered in a dosageof about 0.1 mg/day to 200 mg/day, and preferably, is administered in adosage of about 1 mg/day to 20 mg/day. Esmolol can be administered in adosage of about 150 mcg/day to 100 gm/day, and preferably, isadministered in a dosage of about 500 mg/day to 30 gm/day. Metoprololcan be administered in a dosage of about 1 mg/day to 500 mg/day, andpreferably, is administered in a dosage of about 5 mg/day to 300 mg/day,and preferably, the dosage of beta-2 agonist is about 0.01 mg/kg/day toabout 2.0 mg/kg/day and the corresponding dosage of beta-1 antagonist is15 mg/kg/day to about 300 mg/kg/day.

The appropriate effective therapeutic amounts of the adrenergic beta-2agonist and the adrenergic beta-1 antagonist within the listed rangescan be readily determined by the skilled artisan.

The present invention additionally encompasses methods for preventingheart failure in a subject with a pre-heart failure condition regardlessof cause of the heart failure (e.g. whether from ischemic ornon-ischemic causes) and regardless of the chronicity of the heartfailure (e.g. acute or chronic), comprising administering to the subjecta therapeutically effective amount of an adrenergic beta-2 agonisteither alone or in combination with a therapeutically effective amountof a beta-1 antagonist. As used herein, “pre-heart failure condition”refers to a condition prior to heart failure. The subject with apre-heart failure condition has not been diagnosed as having heartfailure, but nevertheless may exhibit some of the typical symptoms ofheart failure and/or have a medical history likely to increase thesubject's risk to developing heart failure.

The invention further provides methods for treating or preventing heartfailure in a subject post myocardial infarction, comprisingadministering to the subject a therapeutically effective amount of anadrenergic beta-2 agonist either alone, or in combination with atherapeutically effective amount of a beta-1 antagonist. As used in thepresent invention, “myocardial infarction” refers to the medical termfor heart attack. Myocardial infarction occurs when the blood supply toan area of the heart is interrupted because of narrowed or blocked bloodvessels. This can cause permanent damage to the heart muscle. Commonsymptoms include substernal, crushing chest pain that may radiate to thejaw or arms. Chest pains may be associated with nausea, sweating andshortness of breath.

“Postmyocardial infarction syndrome”, as used herein, refers to thecomplications of myocardial infarction (heart attack) such as fever,chest pain, and pericarditis (inflammation of the sac surrounding theheart). Preinfarction syndrome refers to the onset of unstable angina(chest pain that leads to a heart attack).

As used herein, “heart tissue degeneration” means a condition ofdeterioration of heart tissue, wherein the heart tissue changes to alower or less functionally-active form. As described above, heart tissuedamage or degeneration may be caused by, or associated with, a varietyof disorders, conditions, and factors, including, without limitation,chronic heart damage, chronic heart failure, acute heart damage, acuteheart failure, injury and trauma, cardiotoxins, radiation, oxidativefree radicals, decreased blood flow, and myocardial infarction.Preferably, the heart tissue degeneration of the present invention wascaused by myocardial infarction or heart failure.

The present invention provides compositions and methods for treating andpreventing heart failure resulting from both ischemic and non-ischemiccauses. As used herein, ischemia (ischaemia) refers to an insufficientblood supply to any part of the body.

The invention also provides methods for treating or preventing heartfailure in a patient status post myocardial infarction, comprisingadministering to the subject a therapeutically effective amount of anadrenergic beta-2 agonist in combination with a therapeuticallyeffective amount of a beta-1 antagonist. These methods encompass, inparticular, methods for reversing damage to the heart immediatelyfollowing myocardial infarction using an adrenergic beta-1 blocker suchas metoprolol in combination with an adrenergic beta-2 agonist such asclenbuterol. In an embodiment, the present invention treats or preventsheart failure by treating or preventing heart tissue degeneration orreversing the effects of heart failure through normalization of calciumhomeostasis. As used herein, “status post myocardial infarction” refersto the condition of a subject closely following occurrence of myocardialinfarction. As described above, the combination of a beta-2 agonist anda beta-1 antagonist can be administered concurrently, sequentially oralternately. In a preferred embodiment, the beta-2 agonist isclenbuterol and the beta-1 antagonist is metoprolol.

The following examples illustrate the present invention, and are setforth to aid in the understanding of the invention, and should not beconstrued to limit in any way the scope of the invention as defined inthe claims which follow thereafter.

EXAMPLE 1 Patient with CHF Treated with Clenbuterol

A patient with New York Heart Association Class III CHF with an ICD,currently taking a heart failure medical regimen, including carvedilol(a non-selective beta-blocking agent), lisinopril (an ACE inhibitor),digoxin and spiranolactone, has shortness of breath on moderate exertionand a peak oxygen consumption of 14 ml/kg/min during cardiopulmonaryexercising testing. After changing from carvedilol to an equivalent doseof metoprolol sustained-release (a selective beta adrenergic blocker),clenbuterol hydrochloride is initiated at a dose of 20 mcg b.i.d. andup-titrated after one week to 40 mcg b.i.d. After six weeks on thiswell-tolerated dose, cardiopulmonary exercise testing is repeated anddemonstrates an increased peak oxygen consumption of 17 ml/kg/day.

EXAMPLE 2 Clenbuterol Improves Cardiac Function

This example studies the therapeutic effects of clenbuterol on chronicheart failure, and determined its underlining mechanisms in patients notsupported by LVAD.

Chronic heart failure was induced by coronary artery ligation in rats,and was confirmed by echocardiography, three weeks post-surgery. Threegroups of rats were studied: (1) Chronic heart failure withoutclenbuterol (n=7); (2) chronic heart failure+clenbuterol (2 mg/kg/day;n=5); and (3) rats after sham operation (n=11). After 8 weeks of oralclenbuterol therapy, echocardiography and direct hemodynamic monitoringwere performed. Rat hearts were then harvested for ex vivo leftventricular pressure-volume relationship (LVPVR) tracings, histologic(trichrome) sections, and molecular assays. Western analysis formyocardial calcium-handling proteins (ryanodine receptors (RyR),SERCA2a, phospholamban, and calcium-sodium exchanger) was assessed bydensitometry.

Rats with coronary artery ligation developed chronic heart failure, ascompared with Sham rats, as was evidenced by decreased left ventricular(LV) pressure (94±2.8 mmHg vs. 114±3.3 mmHg), LVdP/dt (2,570±384 mmHg/svs. 3,728±193 mmHg/s), and LV fractional area change (35±3% vs. 53±2%),and elevated LVEDP (27±5 mmHg vs. 12±3 mmHg) (all p<0.05).Clenbuterol-treated chronic heart failure rats had significantlyimproved hemodynamic parameters (LV pressure: 100±6 mmHg; LVEDP: 15±6mmHg; LVdP/dt: 3,250±325 mmHg/s) and fractional area change (42±1%) (allp<0.05), as compared to chronic heart failure rats without clenbuterol.There was a left-shift in the LVPVR (a lower volume for a given LVpressure=15 mmHg: 1.02 mlat 15 mmHg vs. 1.11 mlat 15 mmHg, p<0.05) fromclenbuterol-treated chronic heart failure rats vs. chronic heart failurerats alone. Histology confirmed comparable infarct sizes in these twogroups. Protein levels of RyR (1.6±0.4 vs. 2.8±0.8, p<0.05) and SERCA2a(1.7±0.5 vs. 2.2±0.4, p<0.05) were decreased in chronic heart failurerats, and recovered (RyR: 2.2±0.7, SERCA2a: 2.0±0.2, p<0.05) inclenbuterol-treated chronic heart failure rats.

These results indicate that clenbuterol improves cardiac function andleft ventricular pressure-volume relationship (LVPVR) in subjectssuffering from chronic heart failure. It is believed that the underlyingmechanism may involve reverse remodeling via the normalization ofcalcium homeostasis.

EXAMPLE 3 Combination Therapy of Clenbuterol and Metoprolol

The benefits of a combined therapy of adrenergic beta-1 blockers andadrenergic beta-2 agonists for treating heart failure can bedemonstrated in a rat ischemic heart failure model. Specifically, acombination of clenbuterol and nietoprolol can be used to treat ratssuffering from ischemic heart failure.

Heart failure can be induced by coronary artery ligation in rats, andconfirmed by echocardiography. Five groups of rats can then be studied:(1) heart failure without clenbuterol or metoprolol; (2) heartfailure+clenbuterol (2 mg/kg/day) and metoprolol (200 mg/kg/day); (3)heart failure+metoprolol alone; (4) heart failure+clenbuterol alone; and(5) rats after sham operation. After approximately 8 weeks of oraltherapy, echocardiography and direct hemodynamic monitoring can beperformed. Rat hearts are harvested for ex vivo left ventricularpressure-volume relationship (LVPVR) tracings, histologic (trichrome)sections, and molecular assays. Western analysis for myocardialcalcium-handling proteins (ryanodine receptors (RyR), SERCA2a,phospholamban, and calcium-sodium exchanger) can be assessed bydensitometry.

It is expected that the results of this study will demonstrate thegeneral benefits of a combined therapy of adrenergic beta-1 blockers andbeta-2 agonists for heart failure. Rats with coronary artery ligationshould develop heart failure, as compared with Sham rats, evidenced bydecreased left ventricular (LV) pressure, LVdP/dt, and LV fractionalarea change, and elevated LVEDP. Accordingly, rats treated with acombination of clenbuterol and metoprolol will demonstrate the mostimproved hemodynamic parameters and fractional area change, as comparedto heart failure rats treated with either clenbuterol alone ormetoprolol alone, and heart failure rats receiving neither clenbuterolor metoprolol.

EXAMPLE 4 Treat and Prevent Heart Failure with Combination Therapy

The benefits of a combined therapy of adrenergic beta-1 blockers andadrenergic beta-2 agonists for treating rats post myocardial infarctioncan also be demonstrated in rats in a post myocardial infarctioncondition. Specifically, a combination of clenbuterol and metoprolol canbe used to treat and prevent heart failure, and reverse damage to theheart in rats with post myocardial infarction condition.

Myocardial infarction can be induced in rats, and confirmed byechocardiography. Five groups of rats can then be studied: (1)post-myocardial infarction without clenbuterol or metoprolol; (2)post-myocardial infarction+clenbuterol (2 mg/kg/day) and metoprolol (200mg/kg/day); (3) post-myocardial infarction+metoprolol alone; (4)post-myocardial infarction+clenbuterol alone; and (5) rats after shaminduction of myocardial infarction. After approximately 8 weeks of oraltherapy, echocardiography and direct hemodynamic monitoring can beperformed. Rat hearts are harvested for ex vivo left ventricularpressure-volume relationship (LVPVR) tracings, histologic (trichrome)sections, and molecular assays. Western analysis for myocardialcalcium-handling proteins (ryanodine receptors (RyR), SERCA2a,phospholamban, and calcium-sodium exchanger) can be assessed bydensitometry.

It is expected that the results of this study will demonstrate thegeneral benefits of a combined therapy of adrenergic beta-1 blockers andbeta-2 agonists preventing heart failure in rats post-myocardialinfarction. Myocardial infarction induced rats should develop heartfailure or heart failure symptoms, as compared with Sham rats, evidencedby decreased left ventricular (LV) pressure, LVdP/dt, and LV fractionalarea change, and elevated LVEDP. Clenbuterol and metoprolol may have asignificant sygergistic therapeutic effect. Accordingly, rats treatedwith a combination of clenbuterol and metoprolol will demonstrate themost improved hemodynamic parameters and fractional area change, ascompared to post-myocardial infarction rats treated with eitherclenbuterol alone or metoprolol alone, and post-myocardial infarctionrats receiving neither clenbuterol or metoprolol.

EXAMPLE 5 Clenbuterol Improves Calcium Homeostasis, Decreases Apoptosis,and Attenuates Diastolic Dysfunction in a Model of IschemicCardiomyopathy

This example shows the use of clenbuterol in an experimental model ofischemic heart failure. The benefit of the β2-adrenergic agonist,clenbuterol (Clen), in LVAD patients with dilated cardiomyopathy hasbeen reported, but its effect on ischemic heart failure (HF) is unknown.This example investigates whether Clen improves cardiac function,induces reverse remodeling, decreases apoptosis, and has synergy with aβ1-antagonist, metoprolol (Meto), in a model of ischemic HF. HF wasinduced by LAD ligation in rats and confirmed by echocardiography 3weeks post-surgery. Rats were randomized to 5 groups: 1) HF withouttherapy; 2) HF+Clen; 3) HF+Meto; 4) HF+Clen+Meto; and 5) rats after shamsurgery. After 9 weeks of therapy, echocardiographic, hemodynamic, andex vivo end-diastolic pressure-volume relationship (EDPVR) measurementswere obtained. Rats with LAD ligation developed HF as compared to Shamrats, with decreased fractional shortening and dP/dtmax and elevatedLVEDP (all p<0.05). Clen-treated ° F. rats had increased weight gain andheart weights (p<0.05 vs HF rats). The Meto-treated group had a lowerheart rate (p<0.01) and LVEDP (p<0.05) vs the HF group. Normalized EDPVRcurves revealed a leftward shift in Clen rats vs Meto and HF. (p<0.05).Clen, Meto, and Clen+Meto groups all had significant decreases in TUNELand 8-oxoG and increased MYH and OGG1 immunohistochemical signals (allp<0.05). Western blot levels of RyR and SERCA2a were decreased in HFrats vs Sham rats and improved in Clen-treated HF. rats. This exampleshows that Clen ameliorates calcium homeostasis, apoptosis, and EDPVRbut does not have synergy with Meto in our model of ischemic HF.

Clenbuterol is a selective β2-adrenergic receptor agonist first used inthe mid-1970s to treat asthma and is approved for use for thisindication in Europe (Salorinne Y, Stenius B, Tukiainen P, Poppius H.Double-blind cross-over comparison of clenbuterol and salbutamol tabletsin asthmatic out-patients. Eur J Clin Pharmacol. 1975; 8:189-95). Thedrug bears close structural similarity to albuterol, differing from thelatter by the presence of chlorine atoms and an amine group in thebenzene ring. These changes enhance its oral absorption and β-2selectivity. Clenbuterol is recognized as a more potent β2-adrenergicagonist than albuterol (Id) and increases muscle bulk to a greaterextent than other β2-agonists in animal models (Carter W J, Lynch M E.Effect of clenbuterol on recovery of muscle mass and carcass proteincontent following experimental hyperthyroidism in old rats. Comp BiochemPhysiol Comp Physiol. 1994; 108:387-94) secondary to an anabolic effectthat is mediated by β2-activation. As a result of its anabolic actions,oral clenbuterol has been used extensively by athletes to enhance musclesize and strength (id. and Muscling in on clenbuterol. Lancet. 1992;340:403.)

Interest in clenbuterol has been recently sparked as a potentialtreatment for cardiac diseases, specifically in regard to improvingcardiac mechanical properties. Petrou, et al found that clenbuteroladministration led to hypertrophy of latissimus dorsi and cardiac musclein rats (Petrou M, Wynne D G, Boheler K R, Yacoub M H. Clenbuterolinduces hypertrophy of the latissimus dorsi muscle and heart in the ratwith molecular and phenotypic changes. Circulation. 1995; 92:11483-9).Clenbuterol promotes cardiac hypertrophy in rats after proximal bandingof the ascending aorta (Wong K, Boheler K R, Petrou M, Yacoub M H.Pharmacological modulation of pressure-overload cardiac hypertrophy:changes in ventricular function, extracellular matrix, and geneexpression. Circulation. 1997; 96:2239-46.) Normal rat hearts treatedwith clenbuterol have also been shown to have elements of “physiologic”hypertrophy, with normal function, morphology, and calcium-handingprotein mRNA levels (Wong K, Boheler K R, Bishop J, Petrou M, Yacoub MH. Clenbuterol induces cardiac hypertrophy with normal functional,morphological and molecular features. Cardiovasc Res. 1998; 37:115-22.).Finally, it was found that clenbuterol improves right ventricularsystolic function after induction of right ventricular failure bybanding of the pulmonary artery in sheep (Hon J K, Steendijk P, PetrouM, Wong K, Yacoub M H. Influence of clenbuterol treatment during sixweeks of chronic right ventricular pressure overload as studied withpressure-volume analysis. J Thorac Cardiovasc Surg. 2001; 122:767-74).

An preliminary report describes the use of clenbuterol (in combinationwith ACE inhibition, β-1 selective blockade, and spironolactone) inpatients with non-ischemic, dilated cardiomyopathy supported with a leftventricular assist device (LVAD) (Yacoub M H. A novel strategy tomaximize the efficacy of left ventricular assist devices as a bridge torecovery. Eur Heart J. 2001; 22:534-540; Yacoub M H, Tansley P, Birks EJ, et al. A novel combination therapy to reverse end-stage heartfailure. Transplant Proc. 2001; 33:2762-4). Ten of 15 patients treatedwith clenbuterol in this study had significant cardiac improvement,allowing for LVAD explantation for recovery (Hon J K, Yacoub M H. Bridgeto recovery with the use of left ventricular assist device andclenbuterol. Ann Thorac Surg. 2003; 75:S36-41). This series represents arate (67%) of myocardial recovery that is more than double that of anypreviously reported study. Still, the effects of clenbuterol on ischemiccardiomyopathy have not been evaluated to date in experimental orclinical studies.

Recent reports show that the toxic effects of β-adrenergic stimulationis mediated primarily via β-1 receptors while β-2 receptor stimulationmay be protective (Lefkowitz R J, Rockman H A, Koch W J. Catecholamines,cardiac beta-adrenergic receptors, and heart failure. Circulation. 2000;101:1634-1637). Myocardial apoptosis has been implicated as a possiblemechanism in the pathogenesis of HF progression (Kang P M, Izumo S.Apoptosis and heart failure: A critical review of the literature. CircRes. 2000; 86: 1107-1113) and has been correlated with the degree ofleft ventricular (LV) remodeling (Abbate A, Biondi-Zoccai G G L, BussaniR, et al. Increased myocardial apoptosis in patients with unfavorableleft ventricular remodeling and early symptomatic post-infarction heartfailure. J Am Coll Cardiol. 2003; 41:753-760). Catecholamine-inducedapoptosis (Zaugg M, Xu W, Lucchinetti E, et al. Beta-adrenergic receptorsubtypes differentially affect apoptosis in adult rat ventricularmyocytes. Circulation. 2000; 102:344-350) and apoptosis inpost-infarction HF (Prabhu S D, Wang G, Luo J, et al. Beta-adrenergicreceptor blockade modulates Bcl-Xs expression and reduces apoptosis infailing myocardium. J Mol Cell Cardiol. 2003; 35:483-393) has been shownto be primarily mediated via β-1 adrenergic receptors. This inventionprovides that the combination of a β-1 blocker, such as metoprolol, andβ-2 agonist, such as clenbuterol, may be synergistic in their effects onHF.

This example utilizes a well-established model of ischemic, chronic HFin rats for this study. The goals are: (1) to examine the effects ofclenbuterol on cardiac function and ventricular remodeling in ischemiccardiomyopathy both alone and in combination with metoprolol, and (2) todetermine the underlining effects of clenbuterol on calcium homeostasisand apoptosis. For this latter objective, the effects of clenbuterolwere evaluated on markers of apoptosis, DNA damage, and DNA repair inour chronic model of HF. In addition, the effects of clenbuterol onprotein expression levels of the ryanodine receptor (RyR) andsarcoplasmic reticulum calcium-ATPase (SERCA_(2a)) were studied.

Methods Laboratory Animals

All studies were performed in compliance with the Guide for the Care andUse of Laboratory Animals (NRC 1996) and were approved by theInstitutional Animal Care and Use Committee of Columbia University.Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass.)weighing 250 to 300 g were used for all experiments. Food and water wereprovided ad libitum, with rats housed in a light- andtemperature-controlled room over the 12-week study period.

Induction of Chronic Heart Failure

After the induction of general anesthesia with the use ofintraperitoneal ketamine (75 mg/kg; Fort Dodge Animal Health, FortDodge, Iowa) and xylazine (5 mg/kg; Lloyd Laboratories, Shenandoah,Iowa), endotracheal intubation with a 16-guage angiocathether wasperformed. Rats were supported by a small animal ventilator (HarvardApparatus, Holliston, Mass.). After performing a left thoracotomy, asham operation (pericardiectomy only) or left anterior descending artery(LAD) ligation with a 7-0 polypropylene suture was performed.Subsequently, a chest tube (16-guage angiocathether) was placed in theleft pleural space prior to closing the incision in three layers.Finally, the chest tube was used to aspirate the pleural cavity andremoved after extubation.

A total of 69 rats were used in this study. LAD ligation surgery wasperformed in 60 rats, of which 37 (62%) survived three weekspost-surgery. Another 9 rats underwent a sham operation (group Sham),with 7 (78%) survivors. Three weeks post-operatively, echocardiographywas performed to establish the baseline level of HF (as measured byfractional shortening). The LAD ligation rats were divided into 4treatment groups matched for the degree of HF and were randomly assignedto one of the following therapies for 9 additional weeks: (1) ratsreceiving no therapy (group HF, n=9); (2) rats receiving high-doseclenbuterol at 1 mg/kg/day (group Clen, n=9); (3) rats receivinghigh-dose metoprolol at 200 mg/kg/day (group Meto, n=9); or (4) ratsreceiving concurrent high-dose clenbuterol at 1 mg/kg/day and high-dosemetoprolol therapy at 200 mg/kg/day (group Clen+Meto, n=10).

Of the 44 surviving rats (37 post-LAD ligation and 7 Sham rats), 39(89%) survived the 12 week follow-up period. There were no differencesin the survival rates among the 5 groups. The final number of ratsincluded: 7 Sham rats, 9 HF rats, 8 Clen rats, 7 Meto rats, and 8Clen+Meto rats. The LAD ligation rats all had a myocardial infarction ofsufficient size to induce HF, spanning≧20% of the LV circumference (asdetermined by trichrome staining).

Oral Pharmacotherapy

Clenbuterol (ICN Biomedicals, Aurora, Ohio) was sonicated andsubsequently dissolved in the drinking water. Metoprolol (Sigma-Aldrich,St. Louis, Mo.) was dissolved in the drinking water either alone or incombination with clenbuterol for the Clen+Meto group. The concentrationsof clenbuterol and metoprolol were varied to keep the study drug dosedelivered within a narrow therapeutic window based on daily waterconsumption. The average dosages of clenbuterol and metoprolol achievedwere 1.1±0.1 mg/kg/day of clenbuterol (Clen group), 198±32 mg/kg/day ofmetoprolol (Meto group), and 1.1±0.1 mg/kg/day of clenbuterol and 232±20mg/kg/day of metoprolol (Clen+Meto group). The treated drinking waterwas made fresh every 48-72 hours. Oral pharmacotherapy was continued fora total of 9 weeks.

Echocardiography

Under mild isoflurane anesthesia, 2-D echo (Sonos-5500, AgilentTechnologies, Palo Alto, Calif.) was performed 3 and 12 weekspost-surgery for baseline (pre-treatment) and post-treatment measures ofcardiac function, respectively. All echocardiography was performed andanalyzed by a single, experienced individual (I.H.) in a blindedfashion. For each echo, LV anteroposterior diameter and short-axis areaat the papillary muscle level were measured to obtain the LVend-diastolic diameter (LVEDD) and area (LVEDA) and end-systolicdiameter (LVESD) and area (LVESA). Fraction shortening (FS) wascalculated as [(LVEDD−LVESD)/LVEDD×100%] and fractional area change(FAC) was calculated as [(LVEDA−LVESA)/LVEDA×100%].

Hemodynamic Measurements

In the terminal experiments 12 weeks post-surgery, the rats wereanesthetized with inhaled isoflurane mixed with oxygen. A 2-FrenchMillar catheter (Millar Instruments, Houston, Tex.) was inserted intothe right carotid artery and pressure measurements collected and savedas the catheter was advanced into the left ventricle. The heart wasquickly arrested in diastole and excised. Hearts were subsequentlyweighed and used for the ex vivo determination of LV end-diastolicpressure-volume relationships (EDPVR). Using Chart 4 (version 4.2.4,ADInstruments, Colorado Springs, Colo.), left ventricular end-diastolicpressure (LVEDP), left ventricular systolic pressure (LVSP), mean aorticpressure (MAoP), heart rate, and maximum and minimum LV dP/dt(dP/dt_(max) and dP/dt_(min)) were later obtained from storedhemodynamic recordings by a single individual (S.X.), who was blinded toanimals' group assignments.

Measurements of Body Weight and Heart Weight

Body weight was measured pre-surgery and 3 and 12 weeks post-surgery.Heart weight was measured immediately after the heart was excised. Theratio of body weight to heart weight was subsequently calculated.

Determination of End-Diastolic Pressure-Volume Relationships

An 18-gauge angiocatheter was placed into the LV through the aorticvalve and connected to a three-way stopcock. A fine hemostat was placedon the atrial side of the mitral annulus to seal the LV. Aftereliminating any air from the system, LV pressures were measured using a5-French Millar micromanometer introduced through the angiocatheter.While recording LV pressure using an analog-to-digital conversionreading system (Chart 4, version 4.2.4; ADInstruments, Colorado Springs,Colo.), saline was infused into the LV cavity in 50 μL increments usinga calibrated 1 mL syringe. The infused fluid was withdrawn and measuredto ensure that no leakage had occurred. Data was utilized only if >95%of the injected volume was recovered. Using commercial software (IgorPro, version 4.0.5.1; WaveMetrics, Lake Oswego, Oreg.), values of LVpressure (P) and volume (V) were fitted according to the equation:

P=βV^(α),

where β is the base constant and α is an index of ventricular stiffness,as previously described (Mirsky I. Assessment of passive elasticstiffness of cardiac muscle: mathetical concepts, physiologic andclinical considerations, directions of future research. Prog CardiovascDis. 1976; 18:277-308). Averaged data were then used to construct themean LVPVR tracings for each group, after normalizing LV volumes fordifferences in heart weight, as reported previously (Rabkin D G, Jia CX, Cabreriza S E, et al. A novel arresting solution for study ofpostmortem pressure—volume curves of the rat left ventricle. J Surg Res.1998; 80:221-8; Amirhamzeh M M, Hsu D T, Cabreriza S E, Jia C X,Spotnitz H M. Myocardial edema: comparison of effects on filling volumeand stiffness of the left ventricle in rats and pigs. Ann Thorac Surg.1997; 63:1293-7) Analyses were performed by a single individual (S.K.),who was blinded to the group assignment. Comparisons between groups weremade based on normalized LV volume measurements at LV pressures of 30mmHg.

Histological Analysis and Tissue Harvest

A short-axis section of the heart at the point of maximal infarction wasfixed in 4% paraformaldehyde solution for 12 hours. Sections were thenembedded in paraffin and 5 μm slices used for trichrome staining. Theinfarct size was determined as a percentage of the LV circumferenceunder light microscopy by a single individual (S.X.) blinded to groupassignment. The remaining heart tissue was flash-frozen in liquidnitrogen and stored at −80° C. for Western blot analysis.

Apoptosis Studies

Histological sections (5 μm thick) were used to performimmunohistochemistry for: terminal deoxynucleotidyltransferase endlabeling (TUNEL; apoptosis marker), 8-oxoG (DNA damage product), MYH(DNA mismatch repair enzyme), and OGG1 (DNA base excision repairenzyme). The percentage of myocytes with nuclear staining was quantifiedin a blinded manner. Because OGG1 is found both in the nucleus andcytoplasm, a scoring system was utilized to semi-quantitatively describethe staining observed: 0=no staining; 1=0 to 15% staining; 2=15 to 30%staining; 3=30 to 50% staining; and 4=>50% staining of the nucleus andcytoplasm.

Western Blot Analysis

Five rat hearts from each group (HF, Clen, Meto, Clen+Meto, and Sham)were randomly selected and used for analysis of calcium-handling proteinexpression levels. Lysates of LV tissue were obtained with the use of ahomogenizer (Brinkmann Instruments, Westbury, N.Y.). Approximately 150mg of heart tissue was placed in a seven-fold volume of lysis buffer (20mM/L Na-HEPES, 4 mM/L EGTA, 1 mM/L DTT, pH 7.4) in the presence ofproteinase inhibitors (0.1 mM/L leupeptin and 0.3 mM/L PMSF). Theprotein concentration was determined using a protein assay kit (Bio-RadLaboratories, Hercules, Calif.). Samples (50 μg) were denatured at 95°C. and size-fractionated using SDS-polyacrylamide gel electrophoresis(PAGE) under reducing conditions. SDS-PAGE was performed using 7.5%separating and 5% stacking gels for SERCA_(2a) and 5% separating and 4%stacking gels for RyR. Electrophoresis was performed in a Miniprotean IIcell (Bio-Rad Laboratories, Hercules, Calif.) followed by transfer ofproteins (using 34 V overnight at 4° C.) onto nitrocellulose in a minitrans-blot transfer cell (Bio-Rad Laboratories, Hercules, Calif.) filledwith transfer buffer (25 m/L Tris-HCl, pH 8.3, 192 mM/L glycine and 20%methanol).

Blots were blocked overnight at 4° C. in 5% nonfat milk diluted in TBS-T(20 mM/L Tris-HCl, pH 7.6 and 137 mM/L NaCl with 0.1% Tween-20). Blotswere then incubated with primary antibody diluted in TBS-T(anti-SERCA_(2a): 1:1,000, ABR Affinity BioReagents, Golden, Colo.;anti-RyR: 1:2,500, gift from Dr. Andrew Marks' laboratory; anti-tubulin:1:1,000, Sigma-Aldrich) for 1 hour at room temperature. After washing inTBS-T, blots were incubated in the presence of a horseradish,peroxidase-labeled secondary antibody (SERCA_(2a): anti-mouse IgG,Amersham Biosciences, Piscataway, N.J.; RyR: anti-rabbit IgG, AmershamBiosciences) diluted 1:4,000 for 40 minutes at room temperature. Blotswere washed again with TBS-T and then developed using ECL reagent(Amersham Biosciences), followed by autoradiography.

Optical densities of protein level signals were quantified with the useof a laser scanning densitometer (Molecular Dynamics, Palo Alto, Calif.)in a blinded manner. SERCA_(2a) and RyR protein levels were expressedrelative to levels of tubulin.

Statistical Analysis

All statistical analysis was performed using SPSS 11.5 software (SPSS,Chicago, Ill.). Comparisons between treatment groups for hemodynamicdata were made using a 2-way analysis of variance (ANOVA), with thegroup and infarct size [categorized as a large (≦30% of LVcircumference) or small infarct (>30% of LV circumference)] as fixedfactors. Comparisons of paired echocardiographic and body weight datafrom pre-treatment and post-treatment timepoints were performed with theuse of repeated measures ANOVA, with the group, infarct size, andtimepoint as fixed factors. Tukey's ad hoc tests were used for allcomparisons between groups. A p-value less than 0.05 was consideredstatistically significant. All data are expressed as a mean±the standarddeviation (SD).

Results Histological Analysis

There were no significant differences in size of the LV infarct, asexpressed as a percentage of the LV circumference, among the groupsundergoing LAD ligation (see FIG. 1). Adequate LV infarctions wereattained after all LAD ligations, with the range of infarct sizesspanning from 20 to 63% of the LV circumference (mean: 38.1±12.2%).

Echocardiographic Data

FIG. 2 depicts the short-axis echocardiographic data for the 5 groups at3 and 12 weeks after surgery. At 3 weeks post-surgery, there was asignificantly lower FS (17.4±5.22) and FAC (29.4±6.20) in the 4 LADligation groups, as compared to Sham rats (FS: 50.8±6.78; FAC:72.3±6.89). There were no differences in the FS or FAC among the 4 LADligation groups prior to oral pharmacotherapy. After 9 weeks of therapy,there were again no differences in the FS or FAC among any of thegroups.

Body and Heart Weights

The body weight and heart weight data are shown in Table 1. Thepercentage change in the body weight was significantly higher in theSham rats, as compared to all the LAD ligation groups. Treatment withclenbuterol alone and in combination with metoprolol, however, led tosignificantly higher increases in body weight, as compared to thecontrol HF group. For heart weight, Sham rats had lower weights than allthe LAD ligation rats. Clenbuterol-treated rats had significantly higherheart weights than both control HF and metoprolol-treated animals.

TABLE 1 Body and heart weight data. HF Clen Meto Clen + Meto Sham % ΔWeight 33.8 ± 4.79*** 49.9 ± 7.18** † 32.3 ± 7.16*** 55.3 ± 9.91* § †76.1 ± 19.3 Heart Wt (gm) 2.16 ± 0.33*    2.64 ± 0.41*** § † 2.17 ±0.34*  2.42 ± 0.32***  1.72 ± 0.09 Notes: Values are expressed as themean ± SD. % Δ weight represents the percentage change in pre-treatmentto post-treatment weights. *p < 0.05 vs Sham. **p < 0.01 vs Sham. ***p <0.001 vs Sham. † p < 0.01 vs. HF. § p < 0.05 vs. Meto. HF = heartfailure, Clen = clenbuterol, Meto = metoprolol, Wt = weight.

Hemodynamic Data

Table 2 depicts the direct hemodynamic data obtained in the studyanimals after 9 weeks of oral pharmacotherapy. Metoprolol-treatedanimals had a significantly lower heart rate than control HF rats. ForLVEDP, while the control HF, Clen, and Clen+Meto group had asignificantly higher LVEDP than Sham rats, the Meto rats were nodifferent from Sham rats and had a lower LVEDP than HF rats (see FIG.3). There were no differences in the systolic or mean LV or aorticpressures among the groups.

TABLE 2 Hemodynamic Data. HF Clen Meto Clen + Meto Sham HR (bpm) 302 ±19.1  281 ± 11.2   243 ± 19.4 ‡ 275 ± 18   291 ± 52.2 LVEDD (mmHg)  22.4± 11.6***  21.1 ± 11.0**   11.1 ± 5.29 †  20.1 ± 9.53** 5.92 ± 1.85 LVSP(mmHg) 113 ± 10.3  109 ± 10.7  107 ± 9.27  108 ± 11.1  116 ± 17.3 MAP(mmHg) 47.2 ± 8.85  44.2 ± 6.06 42.7 ± 5.38 43.4 ± 4.77 39.8 ± 5.56Maximum dP/dt 4609 ± 583** 4933 ± 596* 5462 ± 541  5327 ± 1270 6700 ±1706 (mmHg/s) Minimum dP/dt −3854 ± 563***  3901 ± 694** −4032 ± 830** −4117 ± 1035** −6458 ± 1799  (mmHg/s) Notes: Values are expressed asthe mean ± SD. *p < 0.05 vs Sham. **p < 0.01 vs Sham. ***p < 0.001 vsSham. † p < 0.05 vs HF. ‡ p < 0.01 vs. HF. HF = heart failure, Clen =clenbuterol, Meto = metoprolol, HR = heart rate, LVEDD = leftventricular end-diastolic diameter, LVSP = left ventricular systolicpressure, MAP = mean arterial pressure.

End-Diastolic Pressure-Volume Relationship Tracings

The ex vivo, passive EDPVR curves obtained are shown in FIG. 4, afternormalization for differences in heart weight. There was a rightwardshift for HF, Meto, and Clen+Meto versus Sham rats. In contrast,clenbuterol-treated rats (Volume at 30 mmHg: 0.42 mL/gm of heart weight)had lower passive LV volumes than either Meto or HF rats (Volume at 30mmHg: 0.51 and 0.50 mL/gm of heart weight, respectively) and were nodifferent from Sham rats (0.36 mL/gm of heart weight).

Apoptosis Studies

The quantitative immunohistochemistry staining of apoptosis, DNA damage,and DNA repair markers is shown in Table 3 and representative images areshown in FIG. 5. The staining of TUNEL was significantly increased inall the LAD ligation groups versus the Sham rats, indicating that therewas increased apoptosis even 12 weeks after surgery in these animals.Clen and Meto treatment both alone and in combination led to decreasedlevels of TUNEL staining versus the HF group. Similarly, for 8-oxoGstaining, the control HF, Clen, and Meto groups had increased levels ofthis DNA damage marker than Sham rats, though Clen, Meto, and Clen+Metoanimals had decreased levels versus the HF rats. Clen+Meto treatment hadan additive effect over Clen or Meto therapy alone, as seen bysignificantly decreased levels of DNA damage with the Clen+Meto groupover Clen or Meto alone.

TABLE 3 Quantitative immunohistochemistry staining of apoptosis, DNAdamage, and DNA repair markers. HF Clen Meto Clen + Meto Sham TUNEL (%)21 ± 4* 11 ± 3* † 12 ± 4* † 8.2 ± 2* †  0.5 ± 0.1 8-oxoG (%) 18 ± 4* 10± 2* † 12 ± 3* † 5 ± 2 † ‡ 0.5 ± 0.1 MYH (score) §  1.4 ± 0.5*  2.8 ±0.5* †  2.6 ± 0.4* † 3.2 ± 0.6* †  0.6 ± 0.2 OGG1 (%)  0.5 ± 0.1*  8.2 ±2.2* †  6.4 ± 1.5* † 13 ± 4* † ‡ 0.2 ± 0.1 Notes: Values are expressedas the mean ± SD. *p < 0.05 vs Sham. † p < 0.05 vs HF. ‡ p < 0.05 vsClen and vs Meto. § scoring system for MYH nuclear and cytoplasmicstaining: 0 = 0%; 1 = 0-15%; 2 = 15-30%; 3 = 30-50%; and 4 = >50%positive. Clen = clenbuterol, M = metoprolol, HF = heart failure.

For the MYH and OGG1 (markers of DNA repair), there were increasedlevels of both markers versus the Sham group for all the LAD ligationgroups. Clen and Meto treatment led to improvements both alone and incombination versus the control HF group. Interestingly, Clen+Metotreatment led to additive improvement in the OGG1 staining pattern overeither Clen or Meto therapy alone. In this study, there was no synergynoted in the effects of Clen+Meto therapy in any of the apoptosis, DNAdamage, or DNA repair marker staining patterns.

Western Blot Analysis

FIG. 6 depicts the calcium-handling protein expression levels for theRyR and SERCA_(2a). The HF rats had significantly decreased levels ofboth RyR and SERCA_(2a) versus Sham rats. These levels weresignificantly improved with the Clen group versus the HF group, andthese levels were not different from those of the Sham group.

Discussion

This is the first study to date to evaluate the effects of the β-2adrenergic agonist, clenbuterol, in ischemic cardiomyopathy. In thismodel of chronic, ischemic HF, clenbuterol treatment led to clearimprovements in calcium homeostasis, diastolic function, myocardialapoptosis and DNA repair. In this study, no evidence was found ofsynergy in the use of this β-2 agonist and a selective β-1 antagonist,metoprolol.

Based on our echocardiographic and histological data, we were able toachieve a significant degree of HF in the experimental model. The sizeof the LV infarction attained was reproducible and uniform across thetreatment arms. In addition, the FS and FAC on echocardiographydemonstrated a significant decrease in LV systolic function in ourchronic model of HF. Finally, repeat echocardiographic and directhemodynamic data at 3 months demonstrated both systolic and diastolicdysfunction in the control HF group verus Sham rats.

High-dose clenbuterol treatment led to significant increases in heartweight and body weight over 9 weeks of oral pharmacotherapy. Incontrast, high-dose metoprolol treatment led to both decreased heartrate and LVEDD with oral therapy. Although serum drug levels were notmonitored in our experiment, these findings are strong evidence ofeffective delivery of each therapy. In addition, the improvementsdemonstrated with metoprolol therapy are similar to prior publishedreports in similar experimental rat post-infarction models, which haveshown decreased heart rate and LVEDD (Prabhu S D, Chandrasekar B, MurrayD R, Freeman G L. Beta-adrenergic blockade in developing heart failure:Effects on myocardial inflammatory cytokines, nitric oxide, andremodeling. Circulation. 2000; 101:2103-2109; Yang Y, Tang Y, Ruan Y, etal. Comparison of metoprolol with low, middle, and high doses ofcarvedilol in prevention of postinfarction left ventricular remodelingin rats. Jpn Heart J. 2003; 44:979-988) but no change in dP/dt_(max)(id) with metoprolol treatment.

Clenbuterol treatment led to improved diastolic function in the Clengroup, as seen by a rightward shift in the normalized EDPVR. HF heartsexhibited substantially enlarged LV volumes relative to non-infarctedSham hearts, with a rightward shift of the pressure-volume curve.Clenbuterol therapy, however, caused a reduction in ventricular cavitydilation, shifting EDPVR curves leftward. This suggests that clenbuterolattenuated deleterious post-infarction LV remodeling. In addition, wefound improvements in calcium homeostasis, as seen by the proteinexpression levels of both the RyR and SERCA_(2a). This is consistentwith the prior clinical findings of improved calcium-handling in DCMpatients treated with mechanical support and clenbuterol therapy(Terracciano C M, Harding S E, Adamson D, et al. Changes in sarcolemmalCa entry and sarcoplasmic reticulum Ca content in ventricular myocytesfrom patients with end-stage heart failure following myocardial recoveryafter combined pharmacological and ventricular assist device therapy.Eur Heart J. 2003; 24:1329-39).

This example also demonstrates that clenbuterol therapy led to decreasedmyocardial apoptosis and increased DNA repair, as seen with quantitativeimmunohistochemistry staining of key markers of apoptosis, DNA damage,and DNA repair. These improvements may explain the reduction indiastolic LV dysfunction seen in the study with clenbuterol, asmyocardial apoptosis has been implicated in the LV remodeling process.Inhibition of apoptosis pathways has also been recently shown toattenuate remodeling (Chandrashekhar Y, Sen S, Anway R, Shuros A, AnandI. Long-term caspase inhibition ameliorates apoptosis, reducesmyocardial troponin-I cleavage, protects left ventricular function, andattenuates remodeling in rats with myocardial infarction. J Am CollCardiol. 2004; 43:295-301).

The combination of a β-2 adrenergic agonist, clenbuterol, and a β-1antagonist, metoprolol, did not lead to any evidence of synergy in thisstudy. Although there was evidence of additive improvements in the8-oxoG and OGG1 staining patterns in the Clen+Meto group, indicatingthat clenbuterol and metoprolol led to independent improvements in DNAdamage and repair, respectively, these effects were not synergistic. Itis possible that a synergistic effect would be seen in a different typeof study or experiment.

The dosages used for clenbuterol and metoprolol were high-dose, raisingthe possibility that synergy was not seen with combinational therapybecause of the counteraction of β-2 adrenergic effects with the use ofan imperfectly selective β-1 antagonist. In addition, the mechanisms bywhich clenbuterol attenuated diastolic dysfunction were not elucidatedby this study.

In summary, this example demonstrates that clenbuterol amelioratescalcium homeostasis, myocardial apoptosis, and EDPVR in a model ofischemic HF. These changes did not have synergy with metoprolol therapy.Trials are underway studying the effects of Clen on cardiac recovery.

Additive effects of β-1 adrenergic antagonism and β-2 stimulation onmyocardial apoptotic inhibition and DNA repair in a model of congestiveheart failure (Abstract). Circulation 2004 Suppl.

All publications referenced herein are hereby incorporated in theirentirety. While the foregoing invention has been described in somedetail for purposes of clarity and understanding, it will be appreciatedby one skilled in the art, from a reading of the disclosure, thatvarious changes in form and detail can be made without departing fromthe true scope of the invention in the appended claims.

While the invention has been described in detail with reference tocertain embodiments thereof, it will be understood that the invention isnot limited to these embodiments. Indeed, modifications and variationsare within the spirit and scope of that which is described and claimed.

1. A method for treating a patient suffering from congestive heartfailure (CHF), not supported by a left ventricle assist device (LVAD),which comprises administering clenbuterol to the patient in an effectiveamount.
 2. The method of claim 1, wherein clenbuterol is administered asclenbuterol hydrochloride.
 3. The method of claim 2, wherein clenbuterolhydrochloride is administered as an oral dosage form.
 4. The method ofclaim 3, wherein clenbuterol hydrochloride is administered in dailydoses from about 40 mcg to about 4 mg.
 5. A method for improvingskeletal muscle function in patients suffering from congestive heartfailure, not supported by an LVAD, which comprises administeringclenbuterol to the patient in an effective amount.
 6. The method ofclaim 5, wherein the clenbuterol comprises clenbuterol hydrochloride. 7.The method of claim 6, wherein clenbuterol hydrochloride is administeredas an oral dosage form.
 8. The method of claim 7, wherein clenbuterolhydrochloride is administered in daily doses from about 40 mcg to about4 mg.
 9. A method of improving cardiac function in patients withcongestive heart failure, not supported by an LVAD, which comprisesadministering clenbuterol.
 10. A method of treating congestive heartfailure in patients, not supported by an LVAD, which comprisesadministering clenbuterol in combination with a beta-1 selectiveblocker.
 11. A method of treating congestive heart failure in patients,not supported by an LVAD, which comprises administering clenbuterol withan ICD.
 12. A method of treating congestive heart failure in patientsnot supported by an LVAD, which comprises administering clenbuterol incombination with a beta-1 selective blocker and an ICD.
 13. A method ofimproving muscle strength as measured in terms of maximal strength orstatic fatigue index, which comprises administering clenbuterol topatients with congestive heart failure, not supported by an LVAD.
 14. Amethod of improving peak oxygen consumption, peak work or exerciseduration during cardiopulmonary exercise testing which comprisesadministering clenbuterol to patients with congestive heart failure, notsupported by an LVAD.
 15. A method of improving New York HeartAssociation Functional Class, which comprises administering clenbuterolto patients with congestive heart failure, not supported by an LVAD. 16.A method of improving patient quality of life according to the MinnesotaLiving with Heart Failure Questionnaire which comprises administeringclenbuterol to patients with congestive heart failure, not supported byan LVAD.
 17. A method of lessening symptoms of heart failure, includingshortness of breath or fatigue which comprises administering clenbuterolto patients with congestive heart failure, not supported by an LVAD. 18.A method of decreasing hospitalizations for exacerbations of CHF whichcomprises administering clenbuterol to patients with congestive heartfailure, not supported by an LVAD.
 19. A method of improving survivalfor patients with CHF which comprises administering clenbuterol topatients with congestive heart failure, not supported by an LVAD
 20. Apharmaceutical composition for use in treating or preventing heartfailure comprising a therapeutically effective amount of an adrenergicbeta-2 agonist.
 21. The pharmaceutical composition of claim 20, whereinthe adrenergic beta-2 agonist is selected from the group consisting ofalbuterol, clenbuterol, formoterol, levalbuterol, metaproterenol,pirbuterol, salmeterol and terbutaline.
 22. A pharmaceutical compositionfor use in treating or preventing heart failure comprising atherapeutically effective amount of an adrenergic beta-1 antagonist incombination with a therapeutically effective amount of an adrenergicbeta-2 agonist.
 23. The pharmaceutical composition of claim 22, whereinthe adrenergic beta-1 antagonist is selected from the group consistingof acebutolol, atenolol, betaxolol, bisoprolol, esmolol and metoprolol,and the adrenergic beta-2 agonist is selected from the group consistingof albuterol, clenbuterol, formoterol, levalbuterol, metaproterenol,pirbuterol, salmeterol and terbutaline.
 24. A method for treating orpreventing heart failure in a subject, comprising administering to thesubject a therapeutically effective amount of an adrenergic beta-2agonist.
 25. The method of claim 24, wherein the adrenergic beta-2agonist treats or prevents heart failure by treating or preventingcardiac arrhythmia.
 26. The method of claim 25, wherein the adrenergicbeta-2 agonist treats or prevents heart failure by treating orpreventing heart tissue degeneration or reversing the effects of heartfailure through the normalization of calcium homeostasis.
 27. The methodof claim 26, wherein the heart tissue degeneration results from ischemicor non-ischemic causes and in an acute or chronic condition.
 28. Themethod of claim 26, wherein the heart tissue degeneration results frommyocardial infarction.
 29. The method of claim 24, wherein the effectiveamount of adrenergic beta-2 agonist is from about 0.01 mg/kg/day toabout 2.0 mg/kg/day.
 30. The method of claim 24, wherein the adrenergicbeta-2 agonist is selected from the group consisting of albuterol,clenbuterol, formoterol, levalbuterol, metaproterenol, pirbuterol,salmeterol, and terbutaline.
 31. The method of claim 30, wherein theadrenergic beta-2 agonist comprises clenbuterol.
 32. The method of claim31, wherein the effective amount of clenbuterol comprises from about0.01 mg/kg/day to about 2 mg/kg/day.
 33. A method of treating orpreventing heart failure in a subject, comprising administering to thesubject a therapeutically effective amount of an adrenergic beta-2agonist, in combination with a therapeutically effective amount of anadrenergic beta-1 antagonist.
 34. The method of claim 33, whereinadministration is concurrent.
 35. The method of claim 33, whereinadministration is sequential.
 36. The method of claim 33, whereinadministration is alternate.
 37. The method of claim 33, wherein asynergistic therapeutic effect results.
 38. The method of claim 33,wherein the heart failure is associated with cardiac arrhythmia.
 39. Themethod of claim 33, wherein the heart failure is associated with hearttissue degeneration.
 40. The method of claim 33, wherein the hearttissue degeneration results from ischemic or non-ischemic causes and inan acute or chronic condition.
 41. The method of claim 33, wherein theheart tissue degeneration results from myocardial infarction.
 42. Themethod of claim 33, wherein the heart tissue degeneration results from amyocardial infarction.
 43. The method of claim 33, wherein the effectiveamount of adrenergic beta-2 agonist is from about 0.01 mg/kg/day toabout 2.0 mg/kg/day.
 44. The method of claim 33, wherein the adrenergicbeta-2 agonist is selected from the group consisting of albuterol,clenbuterol, formoterol, levalbuterol, metaproterenol, pirbuterol,salmeterol, and terbutaline, and the adrenergic beta-1 antagonist isselected from the group consisting of acebutolol, atenolol, betaxolol,bisoprolol, esmolol and metoprolol.
 45. The method of claim 33, whereinthe adrenergic beta-2 agonist comprises clenbuterol.
 46. The method ofclaim 45, wherein the effective amount of clenbuterol is from about 0.01mg/kg/day to about 2 mg/kg/day.
 47. The method of claim 33, wherein theadrenergic beta-1 antagonist comprises metoprolol.
 48. The method ofclaim 33, wherein the effective amount of adrenergic beta-1 blocker isfrom about 15 mg/kg/day to about 200 mg/kg/day.
 49. The method of claim44, wherein the effective amount of metoprolol is from about 15mg/kg/day to about 200 mg/kg/day.
 50. A method for preventing heartfailure a subject with a pre-heart failure condition, comprisingadministering to the subject a therapeutically effective amount of anadrenergic beta-2 agonist.
 51. A method for preventing heart failure ina subject with a pre-heart failure condition, comprising administeringto the subject a therapeutically effective amount of a beta-2 antagonistin combination with a therapeutically effective amount of a beta-1antagonist.
 52. A method for treating or preventing heart failure in asubject post myocardial infarction, comprising administering to thesubject a therapeutically effective amount of an adrenergic beta-1agonist.
 53. A method for treating or preventing heart failure in apatient status post myocardial infarction, comprising administering tothe subject a therapeutically effective amount of an adrenergic beta-2agonist.
 54. A method for treating or preventing heart failure in apatient status post myocardial infarction, comprising administering tothe subject a therapeutically effective amount of an adrenergic beta-2agonist in combination with a therapeutically effective amount of beta-1antagonist.
 55. A method of treating or preventing heart failure in asubject, comprising administering to the subject an amount ofclenbuterol effective to treat or prevent the heart failure, incombination with an amount of an adrenergic beta-1 antagonist effectiveto reduce the toxicity of clenbuterol.
 56. A method of preventing hearttissue degeneration by administering to a subject a therapeuticallyeffective amount of an adrenergic beta-1 antagonist in combination witha therapeutically effective amount of an adrenergic beta-2 agonist. 57.A method for reversing damage to the heart resulting from ischemic ornon-ischemic causes, using a combination of an adrenergic beta-1 blockerand an adrenergic beta-2 agonist.
 58. A method for reversing damage tothe heart following myocardial infarction using a combination of anadrenergic beta-1 blocker and an adrenergic beta-2 agonist.
 59. A kitfor use in treating and preventing heart failure comprising acombination of an adrenergic beta-1 blocker and an adrenergic beta-2.60. A kit for use in reversing damage to the heart resulting fromischemic or non-ischemic causes, comprising a combination of anadrenergic beta-1 blocker and an adrenergic beta-2.
 61. A kit for use inreversing damage to the heart following myocardial infarction,comprising a combination of an adrenergic beta-1 blocker and anadrenergic beta-2.